Anti-human vista antibodies and use thereof

ABSTRACT

The invention provides agonistic anti-human VISTA antibodies and antibody fragments. These agonist antibodies and antibody fragments may be used to potentiate or enhance or mimic VISTA&#39;s suppressive effects on T cell immunity and thereby suppress T cell immunity. These agonist antibodies and antibody fragments are especially useful in the treatment of autoimmunity, allergy, inflammatory conditions, GVHD, sepsis and transplant recipients. Screening assays for identifying these agonists are also provided.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos.62/323,193 filed Apr. 15, 2016, 62/343,355 filed May 31, 2016,62/372,362 filed Aug. 9, 2016, 62/385,627 filed Sep. 9, 2016, 62/425,184filed Nov. 22, 2016, 62/363,929 filed Jul. 19, 2016, 62/365,085 filedJul. 21, 2016, 62/385,805 filed Sep. 9, 2016, 62/363,931 filed Jul. 19,2016, 62/365,102 filed Jul. 21, 2016, 62/385,871 filed Sep. 9, 2016,62/363,917 filed Jul. 19, 2016, 62/365,081 filed Jul. 21, 2016,62/385,888 filed Sep. 9, 2016, 62/364,073 filed Jul. 19, 2016,62/365,166 filed Jul. 21, 2016, 62/385,893 filed Sep. 9, 2016,62/363,925 filed Jul. 19, 2016, 62/365,087 filed Jul. 21, 2016,62/385,785 filed Sep. 9, 2016, 62/406,632 filed Oct. 11, 2016, each andall of which are incorporated herein by reference. This applicationrelates to PCT application PCT/US17/27800 filed Apr. 14, 2017“ANTI-HUMAN VISTA ANTIBODIES AND USE THEREOF” (Attorney Docket No.43260.2213) which is being incorporated by reference and to whichpriority is also claimed.

FIELD

The invention in some embodiments relates to novel anti-human VISTAantibodies and antibody fragments, i.e., anti-human VISTA (k-regionImmunoglobulin-containing Suppressor of T cell Activation(1)), (“VISTA”)antibodies and antibody fragments. More specifically, the presentapplication provides novel human VISTA agonists, i.e., anti-human VISTAantibodies and antibody fragments which agonize or promote thesuppressive effects of human VISTA on immunity, particularly T cellimmunity. Also, the invention relates to the use of such agonists toenhance or mimic the suppressive effects of VISTA on immunity such asits suppressive effects on CD4⁺ or CD8⁺ T cell proliferation, CD4⁺ orCD8⁺ T cell activation and its suppressive effect on the production ofimmune cytokines, particularly proinflammatory cytokines. Also theinvention relates to the specific use of these agonistic antibodies andantibody fragments as prophylactics or therapeutics, especially intreating conditions wherein the prevention or inhibition of T cellimmunity and the expression of proinflammatory cytokines istherapeutically beneficial such as autoimmunity, inflammation, allergicdisorders, sepsis, GVHD or in alleviating the inflammatory side effectsof some conditions such as cancer and more specifically IBD, psoriasis,GVHD, lupus, chronic infection and hepatotoxicity and rheumatoidarthritis.

SEQUENCE LISTING

This application includes as part of its disclosure a biologicalsequence listing text file named “43260o2212.txt” having a size of536,156 bytes that was created Jul. 3, 2017, which is incorporated byreference in its entirety.

BACKGROUND

Immune negative checkpoint regulator (NCR) pathways have proven to beextraordinary clinical targets in the treatment of human immune-relateddiseases. Blockade of two NCRs, CTLA-4 and PD-1, using monoclonalantibodies (mAbs) to enhance tumor immunity is revolutionizing thetreatment of cancer and has established these pathways as clinicallyvalidated targets in human disease. Also soluble versions of NCR ligandsthat trigger NCR pathways have entered the clinic as immunosuppressivedrugs to treat autoimmunity (i.e., AMP-110/B7-H4-Ig for Rheumatoidarthritis).

VISTA (see Ref 1), is an NCR ligand, whose closest phylogenetic relativeis PD-L1. VISTA bears homology to PD-L1 but displays a unique expressionpattern that is restricted to the hematopoietic compartment.Specifically, VISTA is constitutively and highly expressed onCD11b^(high) myeloid cells, and expressed at lower levels on CD⁴⁺ andCD8⁺ T cells. Like PD-L1, VISTA is a ligand that profoundly suppressesimmunity (Ref 1), and like PD-L1, blocking VISTA allows for thedevelopment of therapeutic immunity to cancer in pre-clinical oncologymodels (see Ref 2). Whereas blocking VISTA enhances immunity, especiallyCD8⁺ and CD⁴⁺ mediated T cell immunity, treatment with a soluble Igfusion protein of the extracellular domain of VISTA (VISTA-Ig)suppresses immunity and has been shown to arrest the progression ofmultiple murine models of autoimmune disease.

Clear scientific evidence has shown that VISTA is a ligand that inducesprofound T cell suppression. Numerous antagonistic anti-human VISTAantibodies have been reported by different groups including DartmouthCollege and Jannsen. These antibodies are useful in the treatment ofconditions wherein the suppression of the immunosuppressive effects ofVISTA on T cell immunity is desired such as cancer and infection.However, to the best of the inventors' knowledge no anti-human VISTAantibody or antibody fragment has been previously identified whichagonizes the effects of human VISTA. Such agonistic anti-human VISTAantibodies and antibody fragments would be desirable in treatingconditions wherein the suppression of immunity, particularly T cellimmunity is desired and/or conditions wherein VISTA expression isaberrantly downregulated.

SUMMARY

It is an object of the invention to provide therapeutic and prophylacticmethods for using antibodies and antibody fragments which specificallybind to human VISTA and variants thereof, e.g., chimeric, human,humanized or multispecific anti-human VISTA antibodies whichspecifically bind to human VISTA and which promote or mimic the effectsof human VISTA on immunity.

It is a specific object of the invention to provide therapeutic andprophylactic methods of using an agonistic antibody or antibody fragmentthereof comprising an antigen binding region that specifically binds tohuman VISTA wherein the agonistic antibody or antibody fragment binds tothe same or overlapping epitope as any one of the anti-human VISTAantibodies having the CDR and variable heavy and light polypeptidesshown in FIG. 4.

It is a specific object of the invention to provide therapeutic andprophylactic methods of using an agonistic antibody or antibody fragmentthereof comprising an antigen binding region that specifically binds tohuman VISTA wherein the antibodies or antibody fragments comprisevariable heavy and light sequences having the CDR polypeptides of anyone of the anti-human VISTA antibodies having the sequences shown inFIG. 4.

It is a specific object of the invention to provide therapeutic andprophylactic methods using an agonistic antibody or antibody fragmentthereof comprising an antigen binding region that specifically binds tohuman VISTA wherein the antibodies or antibody fragments comprisevariable heavy and light sequences having the CDR polypeptides of ananti-human VISTA antibody selected from VSTB49-VSTB116.

It is a specific object of the invention to provide therapeutic andprophylactic methods of using an agonistic antibody or antibody fragmentthereof comprising the CDRs of an anti-human VISTA antibody selectedfrom VSTB49-VSTB116, which comprise a variable heavy and/or variablelight polypeptide having at least 90%, 95%, or 96-99% sequence identityto the variable heavy and light polypeptide sequences of VSTB49-VSTB116.

It is a specific object of the invention to provide therapeutic andprophylactic methods of using an agonistic antibody or antibody fragmentthereof comprising the same CDRs any one of VSTB49-VSTB116, whichcomprise a variable heavy and/or variable light polypeptide which is/areidentical to the variable heavy and light polypeptide sequences ofVSTB49-VSTB116.

It is a specific object of the invention to provide therapeutic andprophylactic methods of using an agonistic antibody or antibody fragmentthereof which are chimeric, human, humanized, multispecific (e.g.,bispecific) anti-human VISTA antibodies or antibody fragments comprisingan antigen binding region that specifically binds to human VISTA whichcomprise variable heavy and light sequences having the CDR polypeptidesas any one of the anti-human VISTA antibodies comprising the CDR andvariable heavy and light polypeptides disclosed in FIG. 4.

It is a specific object of the invention to provide therapeutic andprophylactic methods of using novel immunosuppressants, i.e., anti-humanVISTA antibodies and antibody fragments, e.g., those containing humanIgG2 constant domains or IgG2 Fc regions, optionally wherein the FcRbinding capability of the human IgG2 constant domains or IgG2 Fc regionsare maintained or are enhanced compared to the wild-type human IgG2constant domains or IgG2 Fc regions, which agonize, elicit or mimic theeffects of human VISTA on immunity, e.g., its suppressive effects on Tcell activity, differentiation and proliferation and its suppressiveeffects on the expression of proinflammatory cytokines.

It is a specific object of the invention to provide therapeutic andprophylactic methods of using novel immunosuppressive antibodies andantibody fragments which enhance or mimic the suppressive effects ofVISTA on T cell immunity, i.e., which suppress CD4+ or CD8+ T cellproliferation, CD4+ or CD8+ T cell activation and its suppression of theproduction of immune cytokines, particularly proinflammatory cytokinessuch as IL-2, IL-4, IL-6, IL-17, TNF-α, and/or GM-CSF(granulocyte-macrophage colony-stimulating factor), and its promotingeffects on the expression of chemokines or chemoattractants such as KC(keratinocyte chemoattractant) or MIP-2 (Macrophage inflammatory protein2).

It is a specific object of the invention to provide therapeutic andprophylactic methods using novel immunosuppressive or agonisticanti-human VISTA antibodies and antibody fragments of specific epitopicspecificity or which compete for binding to human VISTA with specificanti-human VISTA antibodies.

It is a specific object of the invention to provide therapeutic andprophylactic methods of using novel immunosuppressive or agonisticanti-human VISTA antibodies and antibody fragments of specific epitopicspecificity or which compete for binding to human VISTA with specificanti-human VISTA antibodies which agonize (enhance, elicit or mimic) thesuppressive effects of VISTA on immunity, e.g., its suppressive effectson T cell immunity, i.e., CD4+ or CD8+ T cell proliferation, CD4+ orCD8+ T cell activation, and/or which suppress the production ofproinflammatory immune cytokines such as IL-2, IL-4, IL-6, IL-17, TNF-α,and/or GM-CSF (granulocyte-macrophage colony-stimulating factor), andits promoting effects on the expression of chemokines orchemoattractants such as KC (keratinocyte chemoattractant) or MIP-2(Macrophage inflammatory protein 2).

It is a specific object of the invention to provide therapeutic andprophylactic methods of using agonistic anti-human VISTA antibodies andantibody fragments as prophylactics or therapeutics, especially intreating conditions wherein preventing or inhibiting or reducing immunereactions is therapeutically desirable, and more particularly whereinthe preventing or inhibiting or reducing T cell immunity, or morespecifically CD4+ or CD8+ mediated T cell immunity is therapeuticallybeneficial such as autoimmunity, inflammation, allergic disorders,sepsis, GVHD, and/or in treating transplant or cell therapy recipients,e.g., CAR-T recipients, or in alleviating the inflammatory side effectsof some conditions such as cancer.

It is another specific object of the invention to provide a diagnosticor therapeutic composition comprising a diagnostically ortherapeutically effective amount of an agonist anti-human VISTA antibodyaccording to the invention, e.g., one containing the same CDRs as any ofthe antibodies having the sequences shown in FIG. 4 which is suitablefor use in human therapy, such as an intravenous, subcutaneous orintramuscular administrable composition.

It is another specific object of the invention to provide diagnostic ortherapeutic methods which use an agonist antibody according to theinvention in association with another immune agonist, e.g., a PD-1 orPD-L1 agonist, e.g., wherein the PD-1 or PD-L1 agonist is selected froman anti-PD-1 antibody or antibody fragment, an anti-PD-L1 antibody orantibody fragment, a PD-L1 polypeptide or fragment thereof which may bemonovalent or multimeric, a PD-1 polypeptide or fragment thereof whichmay be monovalent or multimeric, or a complex or fusion proteincomprising any of the foregoing.

It is another specific object of the invention to provide methods ofcontacting immune cells in vitro or in vivo with an agonist antibodyaccording to the invention, e.g., human immune cells, e.g., wherein thecontacted cells are infused into a human subject such as a subject whohas an inflammatory, allergic or autoimmune condition, e.g., GVHD,chronic or acute hepatitis, RA, IBD, psoriasis, or lupus.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated antibody or antibodyfragment thereof comprising an antigen binding region that specificallybinds to human V-domain Ig Suppressor of T cell Activation (humanVISTA), wherein the antibody or antibody fragment agonizes or promotesone or more of the effects of VISTA on immunity, e.g., a human IgG2constant or human IgG2 Fc region optionally wherein the human IgG2constant or Fc region of the antibody binds to Fc gamma receptorsincluding human CD32A, e.g., wherein the IgG2 constant or Fc regioncomprises the native human IgG2 binding to one or more Fc receptors,optionally one or more of hFcγRI (CD64), FcyRIIA or hFcyRIIB, (CD32 orCD32A) and FcγRIIIA (CD16A) or FcγRIIIB (CD16B).

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic antibodyor antibody fragment wherein the isolated antibody or antibody fragmentcompetes with or binds to a VISTA epitope which includes or overlapswith the epitope bound by any of the anti-human VISTA antibodies havingthe sequences of FIG. 4.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic antibodyor antibody fragment wherein the isolated antibody or antibody fragmentcompetes with or binds to a VISTA epitope binds or interacts with one ofmore residues of an epitope comprising residues ofLLDSGLYCCLVVEIRHHHSEHRVH(SEQ ID NO:92).

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic antibodyor antibody fragment wherein the isolated antibody or antibody fragmentcompetes with or binds to a VISTA epitope comprising one or moreresidues of 79EVQTCSERRPIR90 (SEQ ID NO:68), 48NVTLTCRLLGPV60,153HHHSEHRVHGAM164, 52LTCRLLGPV60, 56LLGPVDKGHDVTFYK70,113LAQRHGLESASDHHG127, 153HHHSEHRVHGAM164, 93TFQDLHLHHGGHQAA107,146CLWEIRHHHSEH158, 53TCRLLGPVDKG63, 123SDHHG127 and/or153HHHSEHRVHGAM164.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic antibodyor antibody fragment wherein the isolated antibody or antibody fragmentcompetes with or binds to a VISTA epitope comprising one or moreresidues of 79EVQTCSERRPIR90 (SEQ ID NO:68).

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic antibodyor antibody fragment wherein the isolated antibody or antibody fragmentpromotes or enhances at least one effect of human VISTA on immunity,e.g. its suppressive effect on any one or more of T cell immunity,activation of monocytes, induction of T-cell proliferation; induction orsuppression of cytokine expression, increased survival of monocytes,induction of antibody-dependent cell-mediated cytotoxicity (ADCC) incells-expressing VISTA; and induction of antibody-dependent cellularphagocytosis (ADCP) in cells-expressing VISTA, e.g., wherein theisolated antibody or antibody fragment comprises an antigen bindingregion that specifically binds to human VISTA, wherein the antibody orantibody fragment comprises variable heavy and light sequences havingthe identical CDR polypeptides as any one of the anti-human VISTAantibodies having the CDR and variable heavy and light polypeptidesshown in FIG. 4, and/or comprises the same CDRs as an antibody selectedfrom VSTB49-VSTB116 and/or comprises a variable heavy and/or variablelight polypeptide having at least 90% sequence identity to those of ananti-human VISTA antibody selected from any one of VSTB49-VSTB116,wherein the variable heavy and light polypeptide sequences thereof areshown in FIG. 4 and/or comprises a variable heavy and/or variable lightpolypeptide having at least 95% sequence identity to those of ananti-human VISTA antibody selected from any one of VSTB49-VSTB116,wherein the variable heavy and light polypeptide sequences thereof areshown in FIG. 4 or comprises a variable heavy and/or variable lightpolypeptide having at least 96-99% sequence identity to those of ananti-human VISTA antibody selected from any one of VSTB49-VSTB116 and/orcomprises a variable heavy and/or variable light polypeptide identicalto those of an anti-human VISTA antibody selected from one ofVSTB49-VSTB116, wherein the variable heavy and light polypeptidesequences thereof are shown in FIG. 4 and/or comprises a human constantdomain, e.g., a human constant domain selected from IgG1, IgG2, IgG3 andIgG4, which optionally is modified, e.g., by deletion, substitution oraddition mutations or any combination of the foregoing and/or whereinthe isolated antibody or antibody fragment comprises or is a Fab,F(ab′)2, or scFv antibody fragment.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment wherein the isolated antibody orantibody fragment promotes or enhances at least one of the effects ofhuman VISTA on immunity, e.g., selected from its suppressive effect Tcell immunity, activation of monocytes, suppression of T-cellproliferation; induction or suppression of cytokine expression,increased survival of monocytes, suppression of antibody-dependentcell-mediated cytotoxicity (ADCC) in cells-expressing VISTA; andsuppression of antibody-dependent cellular phagocytosis (ADCP) ofcells-expressing VISTA, e.g., one comprising a human IgG2 constant or Fcregion, e.g., wherein the isolated antibody or antibody fragmentpromotes or enhances the suppressive effect of human VISTA on immunity,e.g. its effect on any one or more of T cell immunity, activation ofmonocytes, T-cell proliferation; cytokine expression, survival ofmonocytes, antibody-dependent cell-mediated cytotoxicity (ADCC) incells-expressing VISTA; and antibody-dependent cellular phagocytosis(ADCP) in cells-expressing VISTA.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment wherein the isolated antibody orantibody fragment inhibits T cell immunity and/or proinflammatorycytokine expression.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment wherein the isolated antibody orantibody fragment comprises a human Fc region, e.g., human IgG1, IgG2,IgG3 and IgG4 or a chimera of any of the foregoing, and/or the isolatedantibody or antibody fragment comprises a chimeric, human, multispecificor humanized antibody or antibody fragment and/or the isolated antibodyor antibody fragment comprises a mutated human IgG2 constant domain orFc region and/or the isolated antibody or antibody fragment comprises ahuman IgG2 constant domain or fragment thereof or an hIgG1, hIgG3,hIgG4, IgA, IgD, IgE, or IgM, wherein the entire or substantially theentire hinge and CH1 domains of said antibody and optionally the entireor substantially the entire light chain constant region have beenreplaced with the corresponding entire or substantially the entire lightchain, and the hinge and CH1 domains (“H2 regions” or “H2 domains”) ofhIgG2 and/or the isolated antibody or antibody fragment (i) comprises anIgG2 Fc region wherein either or both of the heavy chain cysteineresidue at position 127 and the light chain cysteine residue at position214 (wherein numbering is according to Kabat) are deleted or changed toa different amino acid residue, resulting in an increase in theagonistic properties of the resultant modified antibody relative to anantibody wherein these residues are unchanged, (ii) the cysteine residueat position 214 in the H2 region of said antibody is mutated orsubstituted with another amino acid and/or one or more of the cysteineresidues at positions 127, 232 or 233 of the heavy chain are deleted orsubstituted with another amino acid, (iii) it comprises a human IgG2constant domain wherein at least one cysteine residue is deleted orchanged to another amino acid, (iv) it competes with or binds to thesame epitope on human VISTA as VSTB95 (variable heavy and lightsequences shown in FIG. 4).

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment wherein the isolated antibody orantibody fragment:

-   -   comprises the V_(H) CDRs of SEQ ID NO:100, 101 and 102 and the        V_(L) CDRs of SEQ ID NO:103, 104 and 105;    -   comprises the V_(H) CDRs of SEQ ID NO:110, 111 and 112 and the        V_(L) CDRs of SEQ ID NO:113, 114 and 115;    -   comprises the V_(H) CDRs of SEQ ID NO:120, 121 and 122 and the        V_(L) CDRs of SEQ ID NO:123, 124 and 125;    -   comprises the V_(H) CDRs of SEQ ID NO:130, 131 and 132 and the        V_(L) CDRs of SEQ ID NO:133, 134 and 135;    -   comprises the V_(H) CDRs of SEQ ID NO:140, 141 and 142 and the        V_(L) CDRs of SEQ ID NO:143, 144 and 145;    -   comprises the V_(H) CDRs of SEQ ID NO:150, 151 and 152 and the        V_(L) CDRs of SEQ ID NO:153, 154 and 155;    -   comprises the V_(H) CDRs of SEQ ID NO:160, 161 and 162 and the        V_(L) CDRs of SEQ ID NO:163, 164 and 165;    -   comprises the V_(H) CDRs of SEQ ID NO:170, 171 and 172 and the        V_(L) CDRs of SEQ ID NO:173, 174 and 175;    -   comprises the V_(H) CDRs of SEQ ID NO:180, 181 and 182 and the        V_(L) CDRs of SEQ ID NO:183, 184 and 185;    -   comprises the V_(H) CDRs of SEQ ID NO:190, 191 and 192 and the        V_(L) CDRs of SEQ ID NO:193, 194 and 195;    -   comprises the V_(H) CDRs of SEQ ID NO:200, 201 and 202 and the        V_(L) CDRs of SEQ ID NO:203, 204 and 205;    -   comprises the V_(H) CDRs of SEQ ID NO:210, 211 and 212 and the        V_(L) CDRs of SEQ ID NO:213, 214 and 215;    -   comprises the V_(H) CDRs of SEQ ID NO:220, 221 and 222 and the        V_(L) CDRs of SEQ ID NO:223, 224 and 225;    -   comprises the V_(H) CDRs of SEQ ID NO:230, 231 and 232 and the        V_(L) CDRs of SEQ ID NO:233, 234 and 235;    -   comprises the V_(H) CDRs of SEQ ID NO:240, 241 and 242 and the        V_(L) CDRs of SEQ ID NO:243, 244 and 245;    -   comprises the V_(H) CDRs of SEQ ID NO:250, 251 and 252 and the        V_(L) CDRs of SEQ ID NO:253, 254 and 255;    -   comprises the VH CDRs of SEQ ID NO:260, 261 and 262 and the        V_(L) CDRs of SEQ ID NO:263, 264 and 265;    -   comprises the V_(H) CDRs of SEQ ID NO:270, 271 and 272 and the        V_(L) CDRs of SEQ ID NO:273, 274 and 275;    -   comprises the V_(H) CDRs of SEQ ID NO:280, 281 and 282 and the        V_(L) CDRs of SEQ ID NO:283, 284 and 285;    -   comprises the V_(H) CDRs of SEQ ID NO:290, 291 and 292 and the        V_(L) CDRs of SEQ ID NO:293, 294 and 295;    -   comprises the V_(H) CDRs of SEQ ID NO:300, 301 and 302 and the        V_(L) CDRs of SEQ ID NO:303, 304 and 305;    -   comprises the V_(H) CDRs of SEQ ID NO:310, 311 and 312 and the        V_(L) CDRs of SEQ ID NO:313, 314 and 315;    -   comprises the V_(H) CDRs of SEQ ID NO:320, 321 and 322 and the        V_(L) CDRs of SEQ ID NO:323, 324 and 325;    -   comprises the V_(H) CDRs of SEQ ID NO:330, 331 and 332 and the        V_(L) CDRs of SEQ ID NO:333, 334 and 335;    -   comprises the V_(H) CDRs of SEQ ID NO:340, 341 and 342 and the        V_(L) CDRs of SEQ ID NO:343, 344 and 345;    -   comprises the V_(H) CDRs of SEQ ID NO:350, 351 and 352 and the        V_(L) CDRs of SEQ ID NO:353, 354 and 355;    -   comprises the V_(H) CDRs of SEQ ID NO:360, 361 and 362 and the        V_(L) CDRs of SEQ ID NO:363, 364 and 365;    -   comprises the V_(H) CDRs of SEQ ID NO:370, 371 and 372 and the        V_(L) CDRs of SEQ ID NO:373, 374 and 375;    -   comprises the V_(H) CDRs of SEQ ID NO:380, 381 and 382 and the        V_(L) CDRs of SEQ ID NO:383, 384 and 385;    -   comprises the V_(H) CDRs of SEQ ID NO:390, 391 and 392 and the        V_(L) CDRs of SEQ ID NO:393, 394 and 395;    -   comprises the V_(H) CDRs of SEQ ID NO:400, 401 and 402 and the        V_(L) CDRs of SEQ ID NO:403, 404 and 405;    -   comprises the V_(H) CDRs of SEQ ID NO:410, 411 and 412 and the        V_(L) CDRs of SEQ ID NO:413, 414 and 415;    -   comprises the V_(H) CDRs of SEQ ID NO:420, 421 and 422 and the        V_(L) CDRs of SEQ ID NO:423, 424 and 425;    -   comprises the V_(H) CDRs of SEQ ID NO:430, 431 and 432 and the        V_(L) CDRs of SEQ ID NO:433, 434 and 435;    -   comprises the V_(H) CDRs of SEQ ID NO:440, 441 and 442 and the        V_(L) CDRs of SEQ ID NO:443, 444 and 445;    -   comprises the V_(H) CDRs of SEQ ID NO:450, 451 and 452 and the        V_(L) CDRs of SEQ ID NO:453, 454 and 455;    -   comprises the V_(H) CDRs of SEQ ID NO:460, 461 and 462 and the        V_(L) CDRs of SEQ ID NO:463, 464 and 465;    -   comprises the V_(H) CDRs of SEQ ID NO:470, 471 and 472 and the        V_(L) CDRs of SEQ ID NO:473, 474 and 475;    -   comprises the V_(H) CDRs of SEQ ID NO:480, 481 and 482 and the        V_(L) CDRs of SEQ ID NO:483, 484 and 485;    -   comprises the V_(H) CDRs of SEQ ID NO:490, 491 and 492 and the        VL CDR polypeptides of SEQ ID NO:493, 494 and 495;    -   comprises the V_(H) CDRs of SEQ ID NO:500, 501 and 502 and the        VL CDR polypeptides of SEQ ID NO:503, 504 and 505;    -   comprises the V_(H) CDRs of SEQ ID NO:510, 511 and 512 and the        VL CDR polypeptides of SEQ ID NO:513, 514 and 515;    -   comprises the V_(H) CDRs of SEQ ID NO:520, 521 and 522 and the        VL CDR polypeptides of SEQ ID NO:523, 524 and 525;    -   comprises the V_(H) CDRs of SEQ ID NO:530, 531 and 532 and the        VL CDR polypeptides of SEQ ID NO:533, 534 and 535;    -   comprises the V_(H) CDRs of SEQ ID NO:540, 541 and 542 and the        VL CDR polypeptides of SEQ ID NO:543, 544 and 545;    -   comprises the V_(H) CDRs of SEQ ID NO:550, 551 and 552 and the        VL CDR polypeptides of SEQ ID NO:553, 554 and 555;    -   comprises the V_(H) CDRs of SEQ ID NO:560, 561 and 562 and the        V_(L) CDRs of SEQ ID NO:563, 564 and 565;    -   comprises the V_(H) CDRs of SEQ ID NO:570, 571 and 572 and the        V_(L) CDRs of SEQ ID NO:573, 574 and 575;    -   comprises the V_(H) CDRs of SEQ ID NO:580, 581 and 582 and the        V_(L) CDRs of SEQ ID NO:583, 584 and 585;    -   comprises the V_(H) CDRs of SEQ ID NO:590, 591 and 592 and the        V_(L) CDRs of SEQ ID NO:593, 594 and 595;    -   comprises the V_(H) CDRs of SEQ ID NO:600, 601 and 602 and the        V_(L) CDRs of SEQ ID NO:603, 604 and 605;    -   comprises the V_(H) CDRs of SEQ ID NO:610, 611 and 612 and the        V_(L) CDRs of SEQ ID NO:613, 614 and 615;    -   comprises the V_(H) CDRs of SEQ ID NO:620, 621 and 622 and the        V_(L) CDRs of SEQ ID NO:623, 624 and 625;    -   comprises the V_(H) CDRs of SEQ ID NO:630, 631 and 632 and the        V_(L) CDRs of SEQ ID NO:633, 634 and 635;    -   comprises the V_(H) CDRs of SEQ ID NO:640, 641 and 642 and the        V_(L) CDRs of SEQ ID NO:643, 644 and 645;    -   comprises the V_(H) CDRs of SEQ ID NO:650, 651 and 652 and the        V_(L) CDRs of SEQ ID NO:653, 654 and 655;    -   comprises the V_(H) CDRs of SEQ ID NO:660, 661 and 662 and the        V_(L) CDRs of SEQ ID NO:663, 664 and 665;    -   comprises the V_(H) CDRs of SEQ ID NO:670, 671 and 672 and the        V_(L) CDRs of SEQ ID NO:673, 674 and 675;    -   comprises the V_(H) CDRs of SEQ ID NO:680, 681 and 682 and the        V_(L) CDRs of SEQ ID NO:683, 684 and 685;    -   comprises the V_(H) CDRs of SEQ ID NO:690, 691 and 692 and the        V_(L) CDRs of SEQ ID NO:693, 694 and 695;    -   comprises the V_(H) CDRs of SEQ ID NO:700, 701 and 702 and the        V_(L) CDRs of SEQ ID NO:703, 704 and 705;    -   comprises the V_(H) CDRs of SEQ ID NO:710, 711 and 712 and the        V_(L) CDRs of SEQ ID NO:713, 714 and 715;    -   comprises the V_(H) CDRs of SEQ ID NO:720, 721 and 722 and the        V_(L) CDRs of SEQ ID NO:723, 724 and 725;    -   comprises the V_(H) CDRs of SEQ ID NO:730, 731 and 732 and the        V_(L) CDRs of SEQ ID NO:733, 734 and 735;    -   comprises the V_(H) CDRs of SEQ ID NO:740, 741 and 742 and the        V_(L) CDRs of SEQ ID NO:743, 744 and 745;    -   comprises the V_(H) CDRs of SEQ ID NO:750, 751 and 752 and the        V_(L) CDRs of SEQ ID NO:753, 754 and 755;    -   comprises the V_(H) CDRs of SEQ ID NO:760, 761 and 762 and the        V_(L) CDRs of SEQ ID NO:763, 764 and 765;    -   comprises the V_(H) CDRs of SEQ ID NO:770, 771 and 772 and the        V_(L) CDRs of SEQ ID NO:773, 774 and 775;    -   comprises the V_(H) CDRs of SEQ ID NO:780, 781 and 782 and the        V_(L) CDRs of SEQ ID NO:783, 784 and 785;    -   comprises the V_(H) CDRs of SEQ ID NO:790, 791 and 792 and the        V_(L) CDRs of SEQ ID NO:793, 794 and 795;    -   comprises the V_(H) CDRs of SEQ ID NO:800, 801 and 802 and the        V_(L) CDRs of SEQ ID NO:803, 804 and 805;    -   comprises the V_(H) CDRs of SEQ ID NO:810, 811 and 812 and the        V_(L) CDRs of SEQ ID NO: 813, 814 and 815.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment wherein the isolated antibody orantibody fragment:

-   -   comprises the V_(H) polypeptide of SEQ ID NO:106 and the V_(L)        polypeptide of SEQ ID NO:108;    -   comprises the V_(H) polypeptide of SEQ ID NO:116 and the V_(L)        polypeptide of SEQ ID NO:118;    -   comprises the V_(H) polypeptide of SEQ ID NO:126 and the V_(L)        polypeptide of SEQ ID NO:128;    -   comprises the V_(H) polypeptide of SEQ ID NO:136 and the V_(L)        polypeptide f SEQ ID NO:138;    -   comprises the V_(H) polypeptide of SEQ ID NO:146 and the V_(L)        polypeptide of SEQ ID NO:148;    -   comprises the V_(H) polypeptide of SEQ ID NO:156 and the V_(L)        polypeptide of SEQ ID NO:158;    -   comprises the V_(H) polypeptide of SEQ ID NO:166 and the V_(L)        polypeptide of SEQ ID NO:168;    -   comprises the V_(H) polypeptide of SEQ ID NO:176 and the V_(L)        polypeptide of SEQ ID NO:178;    -   comprises the V_(H) polypeptide of SEQ ID NO:186 and the V_(L)        polypeptide of SEQ ID NO:188;    -   comprises the V_(H) polypeptide of SEQ ID NO:196 and the V_(L)        polypeptide of SEQ ID NO:198;    -   comprises the V_(H) polypeptide of SEQ ID NO:206 and the V_(L)        polypeptide of SEQ ID NO:208;    -   comprises the V_(H) polypeptide of SEQ ID NO:216 and the V_(L)        polypeptide of SEQ ID NO:218;    -   comprises the V_(H) polypeptide of SEQ ID NO:226 and the V_(L)        polypeptide of SEQ ID NO:228;    -   comprises the V_(H) polypeptide of SEQ ID NO:236 and the V_(L)        polypeptide of SEQ ID NO:238;    -   comprises the V_(H) polypeptide of SEQ ID NO:246 and the V_(L)        polypeptide of SEQ ID NO:248;    -   comprises the V_(H) polypeptide of SEQ ID NO:256 and the V_(L)        polypeptide of SEQ ID NO:258;    -   comprises the V_(H) polypeptide of SEQ ID NO:266 and the V_(L)        polypeptide of SEQ ID NO:268;    -   comprises the V_(H) polypeptide of SEQ ID NO:276 and the V_(L)        polypeptide of SEQ ID NO:278;    -   comprises the V_(H) polypeptide of SEQ ID NO:286 and the V_(L)        polypeptide of SEQ ID NO:288;    -   comprises the V_(H) polypeptide of SEQ ID NO:296 and the V_(L)        polypeptide of SEQ ID NO:298;    -   comprises the V_(H) polypeptide of SEQ ID NO:306 and the V_(L)        polypeptide of SEQ ID NO:308;    -   comprises the V_(H) polypeptide of SEQ ID NO:316 and the V_(L)        polypeptide of SEQ ID NO:318;    -   comprises the V_(H) polypeptide of SEQ ID NO:326 and the V_(L)        polypeptide of SEQ ID NO:328;    -   comprises the V_(H) polypeptide of SEQ ID NO:336 and the V_(L)        polypeptide of SEQ ID NO:338;    -   comprises the V_(H) polypeptide of SEQ ID NO:346 and the V_(L)        polypeptide of SEQ ID NO:348;    -   comprises the V_(H) polypeptide of SEQ ID NO:356 and the V_(L)        polypeptide of SEQ ID NO:358;    -   comprises the V_(H) polypeptide of SEQ ID NO:366 and the V_(L)        polypeptide of SEQ ID NO:368;    -   comprises the V_(H) polypeptide of SEQ ID NO:376 and the V_(L)        polypeptide of SEQ ID NO:378;    -   comprises the V_(H) polypeptide of SEQ ID NO:386 and the V_(L)        polypeptide of SEQ ID NO:388;    -   comprises the V_(H) polypeptide of SEQ ID NO:396 and the V_(L)        polypeptide of SEQ ID NO:398;    -   comprises the V_(H) polypeptide of SEQ ID NO:406 and the V_(L)        polypeptide of SEQ ID NO:408;    -   comprises the V_(H) polypeptide of SEQ ID NO:416 and the V_(L)        polypeptide of SEQ ID NO:418;    -   comprises the V_(H) polypeptide of SEQ ID NO:426 and the V_(L)        polypeptide of SEQ ID NO:428;    -   comprises the V_(H) polypeptide of SEQ ID NO:436 and the V_(L)        polypeptide of SEQ ID NO:438;    -   comprises the V_(H) polypeptide of SEQ ID NO:446 and the V_(L)        polypeptide of SEQ ID NO:448;    -   comprises the V_(H) polypeptide of SEQ ID NO:456 and the V_(L)        polypeptide of SEQ ID NO:458;    -   comprises the V_(H) polypeptide of SEQ ID NO:466 and the V_(L)        polypeptide of SEQ ID NO:468;    -   comprises the V_(H) polypeptide of SEQ ID NO:476 and the V_(L)        polypeptide of SEQ ID NO:478;    -   comprises the V_(H) polypeptide of SEQ ID NO:486 and the V_(L)        polypeptide of SEQ ID NO:488;    -   comprises the V_(H) polypeptide of SEQ ID NO:496 and the V_(L)        polypeptide of SEQ ID NO:498;    -   comprises the V_(H) polypeptide of SEQ ID NO:506 and the V_(L)        polypeptide of SEQ ID NO:508;    -   comprises the V_(H) polypeptide of SEQ ID NO:516 and the V_(L)        polypeptide of SEQ ID NO:518;    -   comprises the V_(H) polypeptide of SEQ ID NO:526 and the V_(L)        polypeptide of SEQ ID NO:528;    -   comprises the V_(H) polypeptide of SEQ ID NO:536 and the V_(L)        polypeptide of SEQ ID NO:533, 534 and 535;    -   comprises the V_(H) polypeptide of SEQ ID NO:546 and the V_(L)        polypeptide of SEQ ID NO:548;    -   comprises the V_(H) polypeptide of SEQ ID NO:556 and the V_(L)        polypeptide of SEQ ID NO:558;    -   comprises the V_(H) polypeptide of SEQ ID NO:566 and the V_(L)        polypeptide of SEQ ID NO:568;    -   comprises the V_(H) polypeptide of SEQ ID NO:576 and the V_(L)        polypeptide of SEQ ID NO:578;    -   comprises the V_(H) polypeptide of SEQ ID NO:586 and the V_(L)        polypeptide of SEQ ID NO:588;    -   comprises the V_(H) polypeptide of SEQ ID NO:596 and the V_(L)        polypeptide of SEQ ID NO:598;    -   comprises the V_(H) polypeptide of SEQ ID NO:606 and the V_(L)        polypeptide of SEQ ID NO:608;    -   comprises the V_(H) polypeptide of SEQ ID NO:616 and the V_(L)        polypeptide of SEQ ID NO:618;    -   comprises the V_(H) polypeptide of SEQ ID NO:626 and the V_(L)        polypeptide of SEQ ID NO:628;    -   comprises the V_(H) polypeptide of SEQ ID NO:636 and the V_(L)        polypeptide of SEQ ID NO:638;    -   comprises the V_(H) polypeptide of SEQ ID NO:646 and the V_(L)        polypeptide of SEQ ID NO:648;    -   comprises the V_(H) polypeptide of SEQ ID NO:656 and the V_(L)        polypeptide of SEQ ID NO:658;    -   comprises the V_(H) polypeptide of SEQ ID NO:666 and the V_(L)        polypeptide of SEQ ID NO:668;    -   comprises the V_(H) polypeptide of SEQ ID NO:676 and the V_(L)        polypeptide of SEQ ID NO:678;    -   comprises the V_(H) polypeptide of SEQ ID NO:686 and the V_(L)        polypeptide of SEQ ID NO:688;    -   comprises the V_(H) polypeptide of SEQ ID NO:696 and the V_(L)        polypeptide of SEQ ID NO:698;    -   comprises the V_(H) polypeptide of SEQ ID NO:706 and the V_(L)        polypeptide of SEQ ID NO:708;    -   comprises the V_(H) polypeptide of SEQ ID NO:716 and the V_(L)        polypeptide of SEQ ID NO:718;    -   comprises the V_(H) polypeptide of SEQ ID NO:726 and the V_(L)        polypeptide of SEQ ID NO:728;    -   comprises the V_(H) polypeptide of SEQ ID NO:736 and the V_(L)        polypeptide of SEQ ID NO:738;    -   comprises the V_(H) polypeptide of SEQ ID NO:746 and the V_(L)        polypeptide of SEQ ID NO:748;    -   comprises the V_(H) polypeptide of SEQ ID NO:756 and the V_(L)        polypeptide of SEQ ID NO:758;    -   comprises the V_(H) polypeptide of SEQ ID NO:766 and the V_(L)        polypeptide of SEQ ID NO:768;    -   comprises the V_(H) polypeptide of SEQ ID NO:776 and the V_(L)        polypeptide of SEQ ID NO:778;    -   comprises the V_(H) polypeptide of SEQ ID NO:786 and the V_(L)        polypeptide of SEQ ID NO:788;    -   comprises the V_(H) polypeptide of SEQ ID NO:796 and the V_(L)        polypeptide of SEQ ID NO:798;    -   comprises the V_(H) polypeptide of SEQ ID NO:806 and the V_(L)        polypeptide of SEQ ID NO:808; and    -   comprises the V_(H) polypeptide of SEQ ID NO:816 and the V_(L)        polypeptide of SEQ ID NO: 818.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody according to any of the foregoing whichcomprises a human IgG2 constant domain wherein optionally at least onecysteine residue is deleted or changed to another amino acid.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment according to any of the foregoingwherein the isolated antibody or antibody fragment comprises anagonistic anti-human VISTA antibody or antibody fragment which mediatesany one or combination of at least one of the followingimmmunoinhibitory effects: (i) decreases immune response, (ii) decreasesT cell activation, (iii) decreases cytotoxic T cell activity, (iv)decreases natural killer (NK) cell activity, (v) decreases T-cellactivity, (vi) decreases pro-inflammatory cytokine secretion, (vii)decreases IL-2 secretion; (viii) decreases interferon-γ production, (ix)decreases Th1 response, (x) decreases Th2 response, (xi) increases cellnumber and/or activity of regulatory T cells, (xii) increases regulatorycell activity and/or one or more of myeloid derived suppressor cells(MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes,(xiii) increases regulatory cell activity and/or the activity of one ormore of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymalstromal cells, TIE2-expressing monocytes, (xiii) increases M2macrophages, (xiv) increases M2 macrophage activity, (xv) increases N2neutrophils, (xvi) increases N2 neutrophils activity, (xvii) increasesinhibition of T cell activation, (xviii) increases inhibition of CTLactivation, (xix) increases inhibition of NK cell activation, (xx)increases T cell exhaustion, (xxi) decreases T cell response, (xxii)decreases activity of cytotoxic cells, (xxiii) reduces antigen-specificmemory responses, (xxiv) inhibits apoptosis or lysis of cells, (xxv)decreases cytotoxic or cytostatic effect on cells, (xxvi) reduces directkilling of cells, (xxvii) decreases Th17 activity, and/or (xxviii)reduces complement dependent cytotoxicity and/or antibody dependentcell-mediated cytotoxicity, with the proviso that said anti-VISTAantibody or antigen-binding fragment may elicit an opposite effect toone or more of (i)-(xxviii) and optionally is used to treatautoimmunity, allergy, inflammation, transplant or sepsis.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment according to any of the foregoing inorder to treat or prevent rheumatoid arthritis.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment according to any of the foregoing inorder to treat or prevent GVHD.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment according to any of the foregoing inorder to treat or prevent psoriasis.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment according to any of the foregoing inorder to treat or prevent IBD or colitis or another inflammatory orautoimmune intestinal disorder.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment according to any of the foregoing inorder to treat or prevent lupus.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment according to any of the foregoing inorder to treat or prevent chronic or acute infection or inflammation orhepatotoxicity associated therewith, e.g., hepatitis A, B, C, D, E or G.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment according to any of the foregoingwhich method comprises the administration to a subject in need thereofat an agonistic antibody or antibody fragment which effects in vitroand/or in vivo any one or combination of at least one of the followingimmmunoinhibitory effects: (i) decreases immune response, (ii) decreasesT cell activation, (iii) decreases cytotoxic T cell activity, (iv)decreases natural killer (NK) cell activity, (v) decreases T-cellactivity, (vi) decreases pro-inflammatory cytokine secretion, (vii)decreases IL-2 secretion; (viii) decreases interferon-γ production, (ix)decreases Th1 response, (x) decreases Th2 response, (xi) increases cellnumber and/or activity of regulatory T cells, (xii) increases regulatorycell activity and/or one or more of myeloid derived suppressor cells(MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes,(xiii) increases regulatory cell activity and/or the activity of one ormore of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymalstromal cells, TIE2-expressing monocytes, (xiii) increases M2macrophages, (xiv) increases M2 macrophage activity, (xv) increases N2neutrophils, (xvi) increases N2 neutrophils activity, (xvii) increasesinhibition of T cell activation, (xviii) increases inhibition of CTLactivation, (xix) increases inhibition of NK cell activation, (xx)increases T cell exhaustion, (xxi) decreases T cell response, (xxii)decreases activity of cytotoxic cells, (xxiii) reduces antigen-specificmemory responses, (xxiv) inhibits apoptosis or lysis of cells, (xxv)decreases cytotoxic or cytostatic effect on cells, (xxvi) reduces directkilling of cells, (xxvii) decreases Th17 activity, and/or (xxviii)reduces complement dependent cytotoxicity and/or antibody dependentcell-mediated cytotoxicity, with the proviso that said anti-VISTAantibody or antigen-binding fragment may elicit an opposite effect toone or more of (i)-(xxviii) and optionally is used to treatautoimmunity, allergy, inflammation, transplant or sepsis.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment according to any of the foregoing inthe treatment or prevention of allergy, autoimmunity, transplant, genetherapy, inflammation, cancer, GVHD or sepsis, or to treat or preventinflammatory, autoimmune, or allergic side effects associated with anyof the foregoing therewith in a human subject.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment according to any of the foregoingfurther comprising the administration of another immunomodulatoryantibody or fusion protein which is selected from immmunoinhibitoryantibodies or fusion proteins targeting one or more of CTLA4, PD-1,PDL-1, LAG-3, TIM-3, BTLA, B7-H4, B7-H3, VISTA, and/or agonisticantibodies or fusion protein targeting one or more of CD40, CD137, OX40,GITR, CD27, CD28 or ICOS.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment according to any of the foregoingwhich includes assaying VISTA protein by the individual's cells or inbodily fluids prior, concurrent and/or after treatment, e.g., onhematopoietic cells and/or on hematopoietic cells selected from any oneor more of myeloid lineage cells and/or a lymphocytes, monocyte or aneutrophils, T cells, B cells, a natural killer (NK) cells or a naturalkiller T (NKT) cells.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment according to any of the foregoingwherein the agonist anti-human VISTA antibody or fragment comprises thesame CDRs as an antibody selected from VSTB49-VSTB116 and a human IgG2Fc region which optionally may be mutated, optionally wherein the IgG2constant or Fc region retains native FcR binding and/or the ability tobind CD32A and/or the agonist anti-human VISTA antibody or fragmentcomprises an affinity or K_(D) for human VISTA which is 50M or less asdetermined by surface plasmon resonance at 37° C. or the agonistanti-human VISTA antibody or fragment comprises an affinity or K_(D) forhuman VISTA which is 1 nM or less as determined by surface plasmonresonance at 37° C.

It is another specific object of the invention to provide a method ofusing an isolated agonistic anti-human VISTA antibody or antibodyfragment according to any of the foregoing to elicit immunosuppressionin vitro or in vivo.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment according to any of the foregoingwhich is selected from any of the antibodies having the variablesequences of FIG. 4.

It is an object to further provide methods of treating or preventingautoimmunity, allergy and inflammation in a subject in need thereof,e.g., an individual with a an acute or chronic human autoimmune,allergic and inflammatory condition, by the administration of animmunosuppressive or agonistic anti-human VISTA antibody or antibodyfragment wherein said immunosuppressive or agonistic anti-human VISTAantibody or antibody fragment, e.g., wherein said immunosuppressive oragonistic anti-human VISTA antibody or antibody fragment agonizes atleast one of the effects of human VISTA on immunity, e.g., itssuppressive effects on T cell activity, differentiation andproliferation, cytokine levels and B cell immunity.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment according to any of the foregoingwhich is used to treat or prevent lupus or a lupus-like condition orlupus-like symptoms or a method of reversing, stabilizing and reducingthe pathological symptoms associated with lupus or lupus-like conditionsby the administration of an immunosuppressive or agonistic anti-humanVISTA antibody or antibody fragment, e.g., “Systemic LupusErythematosus” or (“SLE”), cutaneous lor skin lupus, drug-induced lupusand neonatal lupus.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment according to any of the foregoingwhich is used to treat or prevent kidney inflammation, inflammatorykidney damage or proteinuria associated with an autoimmune orinflammatory condition in a subject in need thereof by theadministration of an immunosuppressive or agonistic anti-human VISTAantibody or antibody fragment.

It is another specific object of the invention to provide a method oftreating or preventing an autoimmune, allergic or inflammatory conditioncomprising administering to a patient in need thereof a therapeuticallyor prophylactically effective amount of an isolated agonistic anti-humanVISTA antibody or antibody fragment according to any of the foregoingwhich is used to treat or prevent an inflammatory condition involvinginflammation induced splenomegaly or lymphoproliferation in a subject inneed thereof by the administration of an immunosuppressive or agonisticanti-human VISTA antibody or antibody fragment.

It is another specific object of the invention to provide a method ofpromoting IL-9 expression and/or reducing LIX/CXCL5 expression in asubject in need thereof by the administration of an immunosuppressive oragonistic anti-human VISTA antibody or antibody fragment according tothe invention.

It is another specific object of the invention to provide a method oftreating, inhibiting or preventing at least one of pathologic sideeffect of lupus or a lupus-like condition wherein said symptoms includeproteinuria, autoantibodies, increased expression of cytokines and otherfactors associated with inflammation, inflammation of the kidneys, i.e.,lupus nephritis, kidney damage increased blood pressure in the lungs,i.e., pulmonary hypertension, breathing difficulties, Inflammation ofthe nervous system and brain, inflammation in cranial blood vessels,hardening of the arteries or coronary artery disease, skin rash, skinlesions, hair loss or any combination of the foregoing by theadministration of an immunosuppressive or agonistic anti-human VISTAantibody or antibody fragment according to the invention, optionallyfurther including the administration of another drug used for thetreatment of lupus which optionally is selected from corticosteroids,other anti-inflammatory agents, anti-malarial drugs, anticoagulants,ACTH, other immunosuppressants such as methotrexate, cyclophosphamide,and other immunomodulatory antibodies such as belimumab.

It is another specific object of the invention to provide a method forpreventing the development of GvHD complications, e.g., acute or chronicGVHD in a human patient which comprises administering to the humanpatient an immunosuppressive or agonistic anti-human VISTA antibody orantibody fragment according to the invention.

It is another specific object of the invention to provide a method fortreating GvHD complications in a human patient, e.g., acute or chronicGVHD which comprises administering to the human patient animmunosuppressive or agonistic anti-human VISTA antibody or antibodyfragment.

It is another specific object of the invention to provide a method fortreating an organ, tissue or immune cells that are to be transplantedinto a recipient in order to prevent an acute or chronic GvHD responseby contacting said organ, tissue or immune cells that are to betransplanted into a recipient with an immunosuppressive or agonisticanti-human VISTA antibody or antibody fragment, e.g., wherein thetransplanted cells, tissue or organ are allogeneic or xenogeneic, e.g.,allogeneic bone marrow or hematopoietic cells or allogeneic precursorsof bone marrow lineage cells and/or wherein said immunosuppressive oragonistic anti-human VISTA antibody or said antibody fragment isadministered prior, concurrent or after transplant or a combinationthereof and/or wherein the transplant comprises allogeneic cells whichare administered to the patient to treat a malignant or genetic or otherdisease of the blood, e.g., aplastic anemia, myelofibrosis, or bonemarrow failure following chemotherapy and radiation therapy.

It is another specific object of the invention to provide a method forreducing susceptibility to an opportunistic infection in a subject whois a bone marrow transplant recipient, comprising selecting a subjectwho has had an allogeneic bone marrow or hematopoietic stem celltransplant; and administering to the subject a therapeutically effectiveamount of a pharmaceutical composition comprising a VISTA agonistantibody and an effective amount of an antigen of the opportunisticinfection; wherein the pharmaceutical composition and the antigen reducethe susceptibility to the opportunistic infection in the subject,optionally further comprising administering or contacting the transplantcells, tissue or organ with an immunosuppressive drug and/or whichfurther includes the administration or use of another drug whichoptionally is selected from TNF-alpha antagonists, IL-6 antagonists,hydroxychloroquine, corticosteroids, other anti-inflammatory agents,anticoagulants, ACTH, and other immunosuppressants such as methotrexate,cyclophosphamide, sulfasalazine, leflunomide, sodium aurothiomalate,cyclosporin, B cell depleting and inhibitory antibodies and otherimmunomodulatory antibodies.

It is another specific object of the invention to provide a method oftreating or preventing treating or preventing psoriasis or anotherinflammatory skin condition, e.g., one involving the infiltration ofimmune cells, e.g., T cells, or a method of reversing, stabilizing andreducing the pathological symptoms associated with psoriasis or anotherinflammatory skin condition, e.g., one involving the infiltration ofimmune cells by the administration of an immunosuppressive or agonisticanti-human VISTA antibody or antibody fragment according to theinvention, optionally wherein said psoriasis or another inflammatoryskin condition, e.g., one involving the infiltration of immune cells isselected from plaque psoriasis, pustular psoriasis, inverse psoriasis,guttate psoriasis, erythrodermic psoriasis, drug-induced psoriasis, orcomprises plaque psoriasis.

It is another specific object of the invention to provide a method oftreating or preventing inhibiting, reversing, or preventing theinfiltration of CD3+ T cells into a tissue wherein said infiltration isassociated with the pathology of an autoimmune or inflammatory conditionin a subject in need thereof by the administration of animmunosuppressive or agonistic anti-human VISTA antibody or antibodyfragment.

It is another specific object of the invention to provide a method oftreating, inhibiting or preventing at least one of pathologic sideeffect of psoriasis or another inflammatory skin condition wherein saidsymptoms include severe itching, skin plaques, redness, other skindiscoloration or patchiness, rash, skin pustules, skin scaliness, nailpitting or discoloration or any combination of the foregoing by theadministration of an immunosuppressive or agonistic anti-human VISTAantibody or antibody fragment optionally including the administration ofanother drug or used for the treatment of psoriasis or anotherinflammatory skin condition which optionally is selected from IL-12antagonists, IL-17 antagonists, IL-23 antagonists, TNF-alphaantagonists, IL-6 antagonists, hydroxychloroquine, corticosteroids,other anti-inflammatory agents, ACTH, and other immunosuppressants suchas methotrexate, cyclophosphamide, sulfasalazine, leflunomide, sodiumaurothiomalate, cyclosporin, retinoids, vitamin D analogs, ciclosporin,hydroxycarbamide, fumarates such as dimethyl fumarate, and otherimmunomodulatory antibodies.

It is another specific object of the invention to provide a method oftreating or preventing treating or preventing an arthritis orarthritis-like conditions or arthritis and arthritis-like symptoms or amethod of reversing, stabilizing and reducing the pathological symptomsassociated with arthritis and arthritis-like conditions by theadministration of an immunosuppressives or agonistic anti-human VISTAantibody or antibody fragment, e.g., wherein arthritis andarthritis-like conditions are selected from rheumatoid arthritis (“RA”),psoriatic arthritis (“PA”) and osteoarthritis (“OA”).

It is another specific object of the invention to provide a method oftreating or preventing treating or preventing joint inflammation, orjoint pain associated with an autoimmune or inflammatory condition in asubject in need thereof by the administration of an immunosuppressivesor agonistic anti-human VISTA antibody or antibody fragment.

It is another specific object of the invention to provide a method oftreating, inhibiting or preventing at least one of pathologic sideeffect of arthritis or an arthritis-like conditions wherein saidsymptoms include joint damage, joint pain, lung or heart inflammation,low red blood cell count, fever, acute or chronic fatigue, vasculitis,fibrosis such as lung fibrosis, renal amyloidosis, atherosclerosis,myocardial infarction, stroke or any combination of the foregoing by theadministration of an immunosuppressives or agonistic anti-human VISTAantibody or antibody fragment.

It is another specific object of the invention to provide a method forpreventing or treating acute or chronic infection and inflammatoryand/or cytokine responses associated with acute or chronic infection ina human patient which comprises administering to the human patient animmunosuppressive or agonistic anti-human VISTA antibody or antibodyfragment.

It is another specific object of the invention to provide a method forpreventing or treating acute or chronic hepatitis infection andinflammatory and/or cytokine responses associated with acute or chronichepatitis infection in a human patient which comprises administering tothe human patient an immunosuppressive or agonistic anti-human VISTAantibody or antibody fragment.

It is another specific object of the invention to provide a method forpreventing or treating hepatotoxicity or liver damage e.g., associatedwith acute or chronic infection and inflammatory and/or cytokineresponses associated with acute or chronic infection or cirrhosis oralcohol or drug abuse in a human patient which comprises administeringto the human patient an agonistic anti-human VISTA antibody or antibodyfragment, e.g., wherein the treated patient has hepatitis A, B, C, D, Eor G.

It is another specific object of the invention to provide a therapeuticor prophylactic method according to any of the foregoing wherein theagonistic anti-VISTA antibody is administered by a systemic ornon-systemic route mode of administration, e.g., the foregoing claimswherein the agonistic anti-VISTA antibody is administered by injection,topically, inhaled, or orally and/or the agonistic anti-VISTA antibodyis administered by intravenous, subcutaneous, intraarterial,intramuscular, parenteral, spinal or epidermal administration (e.g., byinjection or infusion) and/or the agonistic anti-VISTA antibody isadministered by a systemic or non-systemic route mode of administrationand/or the agonistic anti-VISTA antibody is administered by intravenous,subcutaneous, intraarterial, intramuscular, parenteral, spinal orepidermal administration (e.g., by injection or infusion).

It is another specific object of the invention to provide a therapeuticor prophylactic method, composition or an antibody or antibody fragmentaccording to any of the foregoing wherein the agonistic anti-VISTAantibody is administered in association with another immune agonist,e.g., an agonistic anti-PD-1 antibody or antibody fragment, an agonisticanti-PD-L1 antibody or antibody fragment, an agonistic PD-L1 polypeptideor fragment thereof which may be monovalent or multimeric, an agonisticPD-1 polypeptide or fragment thereof which may be monovalent ormultimeric, or a complex or fusion protein comprising any of theforegoing wherein these agonists may be administered separately or incombination and in either order.

It is another specific object of the invention to provide a therapeuticor prophylactic method or an antibody or antibody fragment according toany of the foregoing wherein the agonistic anti-VISTA antibody is human,humanized, multispecific or chimeric.

It is another specific object of the invention to provide a therapeuticor prophylactic method, composition or an antibody or antibody fragmentaccording to any of the foregoing wherein the agonistic anti-VISTAcomprises human IgG2 constant or Fc regions which optionally may bemutated.

It is another specific object of the invention to provide a therapeuticor prophylactic method or an antibody or antibody fragment according toany of the foregoing wherein the agonistic anti-VISTA antibody comprisesa human IgG2 constant domain or fragment thereof or an hIgG1, hIgG3,hIgG4, IgA, IgD, IgE, or IgM, wherein the entire or substantially theentire hinge and CH1 domains of said antibody and optionally the entireor substantially the entire light chain constant region have beenreplaced with the corresponding entire or substantially the entire lightchain, and the hinge and CH1 domains (“H2 regions” or “H2 domains”) ofhIgG2.

It is another specific object of the invention to provide a therapeuticor prophylactic method or an antibody or antibody fragment according toany of the foregoing wherein the agonistic anti-VISTA antibody comprisesa human IgG2 constant domain or fragment thereof wherein either or bothof the heavy chain cysteine residue at position 127 and the light chaincysteine residue at position 214 (wherein numbering is according toKabat) are deleted or changed to a different amino acid residue,resulting in an increase in the agonistic properties of the resultantmodified antibody relative to an antibody wherein these residues areunchanged and/or the VISTA agonist antibody comprises a human IgG2constant domain or fragment thereof wherein the cysteine residue atposition 214 in the H2 region of said antibody is mutated or substitutedwith another amino acid and/or one or more of the cysteine residues atpositions 127, 232 or 233 of the heavy chain are deleted or substitutedwith another amino acid and/or the VISTA agonist antibody comprises ahuman IgG2 constant domain or fragment thereof wherein at least onecysteine residue is deleted or changed to another amino acid and/or theagonist comprises an antibody having the same CDRs as any of theantibodies in FIG. 4.

It is another specific object of the invention to provide a therapeuticor prophylactic method according to any of the foregoing or an antibodyor antibody fragment according to any of the foregoing wherein theagonistic anti-VISTA antibody competes with or binds to the same epitopeon human VISTA as any of 1E8, GA1, GG8, or any of the other antibodieshaving the sequences shown in FIG. 4.

It is another specific object of the invention to provide a therapeuticor prophylactic method according to any of the foregoing or an antibodyor antibody fragment according to any of the foregoing wherein theagonistic anti-VISTA antibody binds to an epitope on human VISTAcomprising one or more of the residues of the epitope on human VISTAbound by any of 1E8, GA1, GG8, INX800, INX01, INX802, INX900-919 or anyof the other antibodies having the sequences shown in FIG. 4.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-D. This figure shows in vitro and in vivo screening assays whichcan be used to identify suppressive VISTA mAbs. A) Purified T cells wereplated on top of anti-CD3 in the presence of the indicated mAb for 72hours. Proliferation was measured by H3 incorporation. B) PurifiedDO11.10 T cells were stimulated by ISQ pulsed APCs for 6 days in thepresence of the indicated antibody. Proliferation was measured throughuse of CTV dilution dye. C) GVHD was induced by transfer of C57BL/6cells into irradiated BALB/c recipients. Mice were Injected I.P. with200 μg of antibody on day 0, 2 and 4 post transfer and survival wasanalyzed. D) Mice were treated with 10 mpk of the indicated antibody 3hours prior to administration of ConA (15 mpk) and IL-2 was analyzed inplasma at 6 by Luminex.

FIG. 2A-F. This figure shows that agonist VISTA antibodies areimmunosuppressive in multiple models of autoimmune disease. A) NZB/W F1mice were treated 3×/week with either 8G8 or Ham Ig (200 μg) starting at25 weeks until the end of the experiment. “X” denotes time points wherethe control treated group had all been sacrificed. B) Mice were treatedwith 200 μg of antibody 3 hours prior to administration of 15 mg/kg(mpk) of ConA and survival was followed for 80 hours. C) Mice weretreated sequentially with Collagen II mAb followed by LPS and arthritiswas measured by measuring for paw swelling. 8G8 and Ham-Ig wereadministered (200 μg) 3× every other day. D) Imiquimod was applied tothe ear of mice daily. At day 14, 8G8 or Ham-Ig (200 μg) wereadministered every other day and ear thickness was measured withcalipers. E, F) Imiquimod was applied to the backs of mice daily. At day9, mice were euthanized and skin was sectioned & stained for CD3expression by IHC.

FIG. 3. This figure shows the expression of VISTA in WT and hV-KI mice.CD4+ T cells, CD8⁺ T cells, Tregs (CD4⁺ FoxP3⁺), and monocytes, CD11b⁺,Ly6C⁺, Ly6G⁻ were isolated from the lymph nodes of WT and VISTA KI mice,and stained with αVISTA antibodies against mouse or human proteinrespectively.

FIG. 4A-4YY contains the sequences of different anti-human VISTAantibodies including those of INX800, INX801, and INX900-INX919.

FIG. 5 shows the effects of exemplary anti-human VISTA antibodies, i.e.,INX800 and INX801 in a ConA hepatitis model which assesses the effectsthereof on the expression of different cytokines, chemokines andchemoattractants.

FIG. 6 shows the effects of exemplary anti-human VISTA antibodies, i.e.,INX800 and INX801 in an in vivo graft versus host disease (GVHD) animalmodel.

FIG. 7 shows the effects of exemplary agonistic anti-human VISTAantibodies, i.e., INX800 or INX801 on CD3-driven T cell immuneresponses.

FIG. 8 shows the effects of exemplary agonistic anti-human VISTAantibodies, i.e., INX800 or INX801 on the number of specific T cellpopulations or on total T cell numbers.

FIG. 9 compares the effects of exemplary anti-human VISTA antibodies inConA assays and on the expression of select proinflammatory cytokinesand inflammation markers, i.e., IL-2, γ interferon and IL-12p70.

FIG. 10A-C: shows different IgG2 Isoforms. (A) Disulfide shuffling leadsto isoforms A and B, along with the transition for A/B (figure fromZhang, A. et al., 2015). (B) Isoforms are distinguishable by RP-HPLC.(C) Observed RP-HPLC chromatogram for INX901.

FIG. 11: shows chemical enrichment of IgG2 A or B isoforms. (Black line,top) Chromatogram shows a dominant left-most peak defining the B-form.(Red line, bottom) Chromatogram shows a dominant right peak defining theA-form.

FIG. 12: compares INX901 Fc-silent variants with respect to disulfideshuffling. (Top) INX901 on an IgG2 backbone exhibits an expected mixtureof A, A/B, and B isoforms. (Middle) INX901Si on a silent IgG1 backboneexists as a single isoform. (Bottom) INX901HSi possesses an IgG1 silentFc region with a CH1/hinge from IgG2, which enables disulfide shufflingequivalent to native IgG2.

FIG. 13. Biochemically skewed INX901 forms can still reduce cytokineproduction in the MLR. Supernatants from two separate MLRs were analyzedfor cytokine production at the 72-hour time point by Luminex analysis.INX901 parental, A skew and B skew all reduced the production of TNFαand IL-2 in a dose dependent fashion.

FIG. 14. Genetically locked INX901 forms can still reduce cytokineproduction in the MLR, but Fc silent variants cannot. Supernatants fromeach MLR were analyzed for cytokine production at the 72-hour time pointby Luminex analysis. INX901 parental, A lock and B lock all reduced theproduction of TNFα and IL-2 in a dose dependent fashion. The Si and HSivariants, which contain mutations to silence the Fc domain, did notconsistently suppress cytokine production.

FIG. 15. Genetically locked INX908 forms can still reduce cytokineproduction in the MLR, but Fc silent variants cannot. Supernatants fromeach MLR were analyzed for cytokine production at the 72-hour time pointby Luminex analysis. INX908 parental, A lock and B lock all reduced theproduction of TNFα and IL-2 in a dose dependent fashion. The Si and HSivariants, which contain mutations to silence the Fc domain, did notconsistently suppress cytokine production.

FIG. 16. This figure schematically describes the Pepscan® technologyused to identify linear and discontinuous epitopes bound by agonistanti-human VISTA antibodies.

FIG. 17: This figure shows that agonist anti-human VISTA antibodies bindto the same core sequence.

FIG. 18: This figure summarizes the epitope analysis for differentanti-human VISTA antibodies according to the invention.

FIG. 19: This figure shows the epitopes bound by agonist anti-humanVISTA antibodies and further identifies important residues involved inbinding.

FIG. 20: Changes in CD4 T cells in peripheral blood. Absolute numbers in100

of blood (left graph); frequencies of CD45+ cells (center graph);frequencies of CD4+ cells (right graph) (n=8 per group, SEM, statisticunpaired T-test, no equal SD).

FIG. 21: Changes in CD4 T cell activation status in peripheral blood.(n=8 per group, SEM, statistic unpaired T-test, no equal SD) (MFI:median fluorescence intensity).

FIG. 22: INX901 treatment prevents weight loss associated with colitisprogression. (n=8 per group, SEM).

FIG. 23: INX901 treatment prevented colon shortening. (n=8 or 4 pergroup, SEM, statistic unpaired T-test, no equal SD).

FIG. 24: INX901 treatment prevented colitis development. Representativepictures of H&E stained sections of the colon for each mouse group.Magnification: pictures on the top are at 4×, on the bottom at 20×.Arrows indicate areas with abundant inflammatory infiltrates. Note theircomplete absence in the INX901-treated colon sample.

FIG. 25 shows INX901 treatment prevented CD3+ T cell recruitment to thecolon. Representative pictures of CD3 stained sections of the colon foreach mouse group. Magnification: pictures on the top are at 4×, on thebottom at 20×.

FIG. 26: INX901 treatment prevented myeloid (CD11b+) cell recruitment tothe colon. Representative pictures of CD11b stained sections of thecolon for each mouse group. Magnification: pictures on the top are at4×, on the bottom at 20×.

FIG. 27 shows changes in spleen CD4 T cells. Spleens were collected atday 46 (40 days post last antibody dosage) and analyzed by flowcytometry (n=8 or 4 per group, SEM, statistic unpaired T-test, no equalSD).

FIG. 28 shows H&E analysis of skin sections from the IMQD treated mice.The Hamster Ig image is on the left and the 8G8 treated group is on theright.

FIG. 29 shows IHC Analysis of skin sections from the IMQD treated mice.The Hamster Ig image is on the left and the 8G8 treated group is on theright.

FIG. 30 shows a quantitative analysis of skin sections from the IMQDtreated mice for CD3+ cells in the field of view.

FIG. 31 shows a quantitative analysis of immunological populations inthe spleen of Hamster Ig and 8G8 treated mice. Spleens were taken andanalyzed at Day 8.

FIG. 32 shows Luminex analysis of IL-2 from a panel of 32 cytokines fromthe 6-hour time point of mice treated with Control-Ig, INX800 or INX801.Mice were pretreated at −3 hours with each of the indicated antibodies.At time 0, mice were dosed with 15 mg/kg of ConA, and then sacrificedand bled at the 6-hour time point.

FIG. 33 shows Luminex analysis of 32 cytokines from the 6-hour timepoint of mice treated with Control-Ig, INX800 or INX801. Mice werepretreated at −3 hours with each of the indicated antibodies. At time 0,mice were dosed with 15 mg/kg of ConA, and then sacrificed and bled atthe 6-hour time point.

FIG. 34 shows ALC counts from ConA-treated mice from Experiment 14 andnaïve mice. Mice were pretreated at −3 hours with each of the indicatedantibodies at 10 mpk. At time 0, mice were dosed with 15 mg/kg of ConA(Experiment 14) or not dosed at all (naïve), and then bled at 6 hoursfor ALC counts by Flow cytometry

FIG. 35 shows Luminex analysis of serum IL-2 from a panel of 32cytokines at the 6-hour time point of mice treated with Ham-Ig, 8G8 or13F3. Mice were pretreated at −3 hours with each of the indicatedantibodies. At time 0, mice were dosed with 15 mg/kg of ConA, and thensacrificed and bled at the 6-hour time point.

FIG. 36 shows Kaplan Meier curves of the 30 mg/kg ConA treated mice.Mice were pretreated at −3 hours with each of the indicated antibodies.At time 0, mice were dosed with 30 mg/kg of ConA, and then followed forsurvival analysis

FIG. 37 shows IL-2 expression in the plasma from the 6-hour time pointof mice treated with Control-Ig, INX800 or INX903. Mice were pretreatedat −3 hours with each of the indicated antibodies. At time 0, mice weredosed with 15 mg/kg of ConA, and then sacrificed and bled at the 6-hourtime point.

FIG. 38A-B shows IL-2 and MIP-1β expression in the plasma from the6-hour time point of mice treated with Control-Ig, INX800, INX903 or anAntagonist. Mice were pretreated at −3 hours with each of the indicatedantibodies. At time 0, mice were dosed with 15 mg/kg of ConA, and thensacrificed and bled at the 6-hour time point.

FIG. 39 contains the experimental protocol for the CIA arthritis model.

FIG. 40 shows the effects of an agonistic anti-mouse VISTA antibody,8G8, in a collagen-induced arthritis model. As shown treatment wasinitiated at day −2 and subsequently mice were dosed every other day.(n=10 in each group). 8G8 treatment significantly reduced diseaseseverity (interaction term P<0.000005).

FIG. 41 contains the experimental protocol for a CIA arthritis modelexperiment in Example 9.

FIG. 42 shows the CAIA disease progression scoring for the experiment ofExample 8. As shown treatment was initiated at day −2 and subsequentlymice were dosed every other day. (n=10 in each group). 8G8 treatmentsignificantly reduced disease severity (interaction term P<0.000005).

FIG. 43 shows CAIA disease progression scoring. Treatment was initiatedat day −2 and subsequently mice were dosed every other day. (n=9 incontrol group and 8 in INX903 treated group; 1 mouse was removed fromthe control group as it never showed any signs of disease). INX903treatment significantly reduced disease severity (interaction termP=0.01).

FIG. 44 shows CAIA disease progression scoring. Treatment was initiatedat day −2 and subsequently mice were dosed every other day. (n=10 ineach group). 8G8 treatment significantly reduced disease severity(interaction term P<0.0001).

FIG. 45 shows CAIA disease progression scoring with INX800 treatment.Treatment was initiated at day −2 and subsequently mice were dosed everyother day. (n=9 in control group and 8 in INX800 treated group).

FIG. 46 shows CAIA disease progression scoring with INX901 treatment.Treatment was initiated at day −2 and subsequently mice were dosed everyother day. (n=9 in control group and 10 in INX901 treated group).

FIG. 47 shows CAIA disease progression scoring with INX902 treatment.Treatment was initiated at day −2 and subsequently mice were dosed everyother day. (n=9 in control group and 7 in INX902 treated group).

FIG. 48A-B show weights and survival of recipient mice treated with 8G8,13F3, or control Hamster IgG antibodies in acute GvHD disease model.

FIG. 49 shows weights of recipient mice treated with INX anti-VISTAantibodies or control Ig and survival in acute GvHD disease model A:Mean weight loss by group (N=5-8 mice per group) at the peak of disease;B: Mean weight loss by group (N=5-8 mice per group); C: Survival.

FIG. 50A-C shows chimerism and donor T-cell numbers in surviving micetreated with INX901, INX902, INX903 and INX904 or control Ig in acuteGvHD disease model. A) Representative plot of donor (H2Kb, vertical) orrecipient (H2Kd, horizontal) expression in blood CD11b cells in α-humanVISTA treated mice (left panel) or Balb/c control mice (right panel) B)Percentage of donor derived CD11b in the blood of α-human VISTA treatedmice. C) donor derived T cells number in 25 uL of blood in chimericα-human VISTA treated mice or in DDE1 control mice.

FIG. 51A-B shows weights of NSG mice treated with INX901 or control ABin xeno-GvHD disease model. 51A: Means by group (N=6) 51B: weights ofindividual mice. Skull and crossbones indicate mice were either founddead or euthanized at the indicated date.

FIG. 52 shows T-cell expansion in NSG mice treated with INX901 orcontrol AB in xeno-GvHD disease model The figure shows the values in %of total CD45+ cells in the mouse peripheral circulation made up ofhuman CD3+ T-cells.

FIG. 53A-C shows Weights and survival of recipient mice treated with 8G8antibodies or control Hamster IgG in acute GvHD disease model; 53A: Meanweight loss by group (N=8 mice per group); 53B: Individual weight lossby group (N=8 mice per group); and 53C: Survival.

FIG. 54A-B shows weights of recipient mice treated with various doses ofINX902 or control Ig and survival in acute GvHD disease model; 54A showsmean weight loss by group (N=8 mice per group) for INX902 treated miceand 54B shows survival for INX902 treated mice.

FIG. 55A-B shows chimerism in surviving mice treated with various dosesof INX902 or control Ig in acute GvHD disease model; 55A showsPercentage of donor derived CD11b in the blood of INX902 treated miceand 55B shows Donor derived T cells number in 25 uL of blood in INX902treated mice or in DDE1 control mice.

FIG. 56A-D shows weights of recipient mice treated with various doses ofINX903 and INX901 antibodies or control Ig and survival in acute GvHDdisease model; 56A shows Mean weight loss by group (N=8 mice per group)and 56B shows survival for INX903 treated mice; 56C shows Mean weightloss by group (N=8 mice per group) for INX901 treated mice; and 56Dshows survival for INX901 treated mice.

FIG. 57A-B shows chimerism in surviving mice treated with various dosesof INX901 and INX901 or control Ig in acute GvHD disease model; 57A:Percentage of donor derived CD11b in the blood of INX903 treated mice;57B: Percentage of donor derived CD11b in the blood of INX901 treatedmice.

FIG. 58A-C shows weights of recipient mice treated with INX antibodiesor control Ig and survival in acute GvHD disease model; 58A shows meanweight loss by group (N=8 mice per group); 58B individual weight loss bygroup (N=8 mice per group) and 16C survival.

FIG. 59 shows acute GvHD was induced by transfer of T cells and BM fromhV-KI mice into irradiated Balb/c recipients. Mice were tracked fordisease by weight loss, with mice being sacrificed if more than 20% ofthe initial starting weight was lost.

FIG. 60 shows that the agonist anti-VISTA antibody 8G8 delaysproteinuria onset in NZBWF-1 mice. In the experiments 16-week-old femaleNZBWF-1 mice were monitored weekly for proteinuria. Proteinuria valueswere recorded using chemstrips and quantified as mg/dL. At week 32, micewere treated with either 300 ug Control-IgG (black line, n=5) or 300 ug8G8 (red line, n=5) by i.p injection three times a week. At week 33,mice treated with Control-IgG were sacrificed due to poor health.

FIG. 61 shows LIX/CXCL5 and IL-9 levels in the serum of Control-Ig and8G8 treated NZBWF-1 mice were detected. Serum was collected at week 33from Control-IgG (n=5) and 8G8 mice (n=5) and chemokines and cytokineswere assessed on a 32 plex run using Bio-plex 200 Systems and analyzedby Bio Plex manager 6.0 software. Data is shown as the mean+/−SEM andstatistical significance was determined by the unpaired Student t Test.In FIG. 7 **denotes significance (p<0.01) between groups.

FIG. 62 shows that 8G8 reduces proteinuria development in NZBWF-1 mice.In the experiments 22-week-old female NZBWF-1 mice were monitored weeklyfor proteinuria. Proteinuria values were recorded using chemstrips andquantified as mg/dL. On week 28, mice were treated with either 300 ugControl-IgG (black line, n=6) or 300 ug 8G8 (red line, n=6) by i.pinjection three times a week.

FIG. 63 shows that the VISTA agonist 8G8 reduces proteinuria developmentin MRL/pr mice in experiments wherein 15 week old female MRL/Ipr micewere monitored weekly for proteinuria. Proteinuria values were recordedusing chemstrips and quantified as mg/dL. On week 16, mice were treatedwith either 300 ug hamster-Ig (black line, n=8) or 300 ug 8G8 (red line,n=8) by i.p injection three times a week. Data at week 21 were discardeddue to technical problems with the chemstrips. (A) Average proteinuriais shown with standard error bars. (B) Disease incidence at each timepoint was calculated as the percent of mice in each group that exhibitedproteinuria at or greater than 100 mg/dL.

FIG. 64 shows that the VISTA agonist 8G8 (anti-mouse VISTA agonistantibody) reduces splenomegaly in MRL/Ipr mice in experiments whereinspleens were harvested on week 23 from mice were treated with either 300ug Control-Ig/hamster-Ig or 300 ug 8G8 by i.p injection three times aweek. Splenomegaly was observed in Control-Ig treated mice compared to8G8 treated mice. Shown here are representative spleens.

FIG. 65 shows that the VISTA agonist 8G8 (anti-mouse VISTA agonistantibody) reduces lymphoproliferation of cervical lymph nodes in MRL/Iprmice in experiments wherein cervical lymph nodes were harvested on week23 from mice treated with either 300 ug Control-Ig/hamster-Ig or 300 ug8G8 by i.p injection three times a week. Lymphoproliferation wasobserved in Control-Ig treated mice compared to 8G8 treated mice. Shownhere are representative cervical lymph nodes.

FIG. 66A-B shows that 8G8 (anti-mouse VISTA agonist antibody) reducesproteinuria development in MRL/Ipr mice in experiments wherein 9 weekold female MRL/Ipr mice were monitored weekly for proteinuria.Proteinuria values were recorded using chemstrips and quantified asmg/dL. On week 11, mice were treated with either 200 uL PBS (dottedblack line, n=8) or 10 mg/kg hamster-Ig (solid black line, n=8) or 10mg/kg 8G8 (red line, n=8) by i.p injection three times a week. (A)Average proteinuria is shown with standard error bars. (B) Diseaseincidence at each time point was calculated as the percent of mice ineach group that exhibited proteinuria at or greater than 100 mg/dL.

FIG. 67 shows experimental design for the DDE1 transfer experimentreferenced in the example. INX903 (anti-human VISTA agonist Abcontaining VSTB95 antibody variable regions (see FIG. 4) and wild-typehuman IgG2 constant regions). In the experiments treatment wasadministered at days 0, 2, and 6 following DDE1 transfer. At each timepoint, 4 mice per group were analyzed plus 1 naïve mouse. Spleens wereprocessed for flow cytometry, and serum was recovered from cardiac bloodfor detection of anti-dsDNA IgG by ELISA.

FIG. 68 contains results of DDE1 transfer experiments. The resultsindicate that donor and host cell populations are distinguishable bytheir MHC class I alleles. The host B6D2F1 cells express both H-2Kb andH-2Kd, whereas the donor DDE1 cells express only H-2Kb.

FIG. 69A-C contains further results of DDE1 transfer experiments. Thefigure shows that B cells activation during SLE progression is preventedby INX903 treatment. Panel A contains histograms plots of MHCII I^(Ad)expression on recipient B cells. Panel B shows the total number ofrecipient B cells and spleen cells over the course of the experiment andMHC class II IAd MFI on recipient B cell over the course of theexperiment (n=4, SEM). Panel C shows the spleen size at day 14.

FIG. 70 contains further results of DDE1 transfer experiments.Particularly the results in the Figure show that anti-dsDNA autoantibodyproduction in SLE is prevented by INX903 treatment. Anti-dsDNA IgG titerin serum measured by ELISA in naïve (n=2), and HuIgG2 or INX903 treatedmice at D7 and D14 (n=4, SEM).

FIG. 71 contains further results of DDE1 transfer experiments. The datain the Figure shows that shows that there is decreased CD69 expressionat Day 1 in INX903-treated CD4 T cells (n=4).

FIG. 72 contains further results of DDE1 transfer experiments. The datain the figure show that there is a decreased number of CD4 T cells inINX903-treated mice, despite no changes in cell cycle (n=4).

FIG. 73 also shows decreased number of total donor CD4 T cells over timein response to INX903 treatment (n=4, SEM).

FIG. 74A-B: shows that 8G8 reduces proteinuria development in MRL/Iprmice. As shown 15 week old female MRL/Ipr mice were monitored weekly forproteinuria. Proteinuria values were recorded using chemstrips andquantified as mg/dL. On week 16, mice were treated with either 300 ughamster-Ig (black line, n=8) or 300 ug 8G8 (red line, n=8) by i.pinjection three times a week. Data at week 21 were discarded due totechnical problems with the chemstrips. (A) Average proteinuria is shownwith standard error bars. (B) Disease incidence at each time point wascalculated as the percent of mice in each group that exhibitedproteinuria at or greater than 100 mg/dL.

FIG. 75 shows that 8G8 administration reduces splenomegaly in MRL/Iprmice. Spleens were harvested on week 23 from mice were treated witheither 300 ug Control-Ig/hamster-Ig or 300 ug 8G8 by i.p injection threetimes a week. Splenomegaly was observed in Control-Ig treated micecompared to 8G8 treated mice. Shown here are representative spleens.

FIG. 76 shows that 8G8 administration reduces lymphoproliferation ofcervical lymph nodes in MRL/Ipr mice. Cervical lymph nodes wereharvested on week 23 from mice treated with either 300 ugControl-Ig/hamster-Ig or 300 ug 8G8 by i.p injection three times a week.Lymphoproliferation was observed in Control-Ig treated mice compared to8G8 treated mice. Shown here are representative cervical lymph nodes.

FIG. 77 contains experimental results indicating that 8G8 administrationreduces proteinuria development in NZBWF-1 mice.

FIG. 78 contains experimental results indicating that 8G8 administrationdoes not affect immune complexes NZBWF-1 mice.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein may be used inthe invention or testing of the present invention, suitable methods andmaterials are described herein. The materials, methods and examples areillustrative only, and are not intended to be limiting. Thenomenclatures utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques may be usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise.

“Activating receptor,” as used herein, refers broadly to immune cellreceptors that bind antigen, complexed antigen (e.g., in the context ofMHC molecules), Ig-fusion proteins, ligands, or antibodies. Activatingreceptors but are not limited to T cell receptors (TCRs), B cellreceptors (BCRs), cytokine receptors, LPS receptors, complementreceptors, and Fc receptors. For example, T cell receptors are presenton T cells and are associated with CD3 molecules. T cell receptors arestimulated by antigen in the context of MHC molecules (as well as bypolyclonal T cell activating reagents). T cell activation via the TCRresults in numerous changes, e.g., protein phosphorylation, membranelipid changes, ion fluxes, cyclic nucleotide alterations, RNAtranscription changes, protein synthesis changes, and cell volumechanges. For example, T cell receptors are present on T cells and areassociated with CD3 molecules. T cell receptors are stimulated byantigen in the context of MHC molecules (as well as by polyclonal T cellactivating reagents). T cell activation via the TCR results in numerouschanges, e.g., protein phosphorylation, membrane lipid changes, ionfluxes, cyclic nucleotide alterations, RNA transcription changes,protein synthesis changes, and cell volume changes.

“Adjuvant” as used herein, refers to an agent used to stimulate theimmune system and increase the response to a vaccine, without having anyspecific antigenic effect in itself.

“Agonist” herein refers to a molecule, generally an antibody or fusionproteins which enhances or mimics the effects of a specific molecule onimmunity. Generally in the present application this will refer toanti-human VISTA agonist antibodies and antibody fragments which enhanceor mimic the effects of human VISTA on immunity, particularly VISTA'ssuppressive effects on T cell immunity (CD4+ and/or CD8+ T cellimmunity), the expression of proinflammatory cytokines and its effectsof the expression of specific chemokines and chemoattractants.

“Aids in the diagnosis” or “aids in the detection” of a disease hereinmeans that the expression level of a particular marker polypeptide orexpressed RNA is detected alone or in association with one or more othermarkers in order to assess whether a subject has cells characteristic ofa particular disease condition or the onset of a particular diseasecondition or comprises immune dysfunction such as immunosuppressioncharacterized by VISTA expression or abnormal immune upregulationcharacterized by cells having reduced VISTA levels, such as duringautoimmunity, inflammation or allergic responses, e.g., in individualswith chronic and non-chronic diseases.

“Allergic disease,” as used herein, refers broadly to a diseaseinvolving allergic reactions. More specifically, an “allergic disease”is defined as a disease for which an allergen is identified, where thereis a strong correlation between exposure to that allergen and the onsetof pathological change, and where that pathological change has beenproven to have an immunological mechanism. Herein, an immunologicalmechanism means that leukocytes show an immune response to allergenstimulation.

“Amino acid,” as used herein refers broadly to naturally occurring andsynthetic amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified(e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.) Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid (i.e., a carbon that isbound to a hydrogen, a carboxyl group, an amino group), and an R group(e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium.) Analogs may have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

“Anergy” or “tolerance,” or “prolonged antigen-specific T cellsuppression” or “prolonged immunosuppression” as used herein refersbroadly to refractivity to activating receptor-mediated stimulation.Refractivity is generally antigen-specific and persists after exposureto the tolerizing antigen has ceased. For example, anergy in T cells (asopposed to unresponsiveness) is characterized by lack of cytokineproduction, e.g., IL-2. T cell anergy occurs when T cells are exposed toantigen and receive a first signal (a T cell receptor or CD-3 mediatedsignal) in the absence of a second signal (a costimulatory signal).Under these conditions, reexposure of the cells to the same antigen(even if reexposure occurs in the presence of a costimulatory molecule)results in failure to produce cytokines and, thus, failure toproliferate. Anergic T cells can, however, mount responses to unrelatedantigens and can proliferate if cultured with cytokines (e.g., IL-2).For example, T cell anergy can also be observed by the lack of IL-2production by T lymphocytes as measured by ELISA or by a proliferationassay using an indicator cell line. Alternatively, a reporter geneconstruct can be used. For example, anergic T cells fail to initiateIL-2 gene transcription induced by a heterologous promoter under thecontrol of the 5′ IL-2 gene enhancer or by a multimer of the APIsequence that can be found within the enhancer (Kang et al. (1992)Science 257: 1134). Modulation of a costimulatory signal results inmodulation of effector function of an immune cell.

“Antagonist” herein refers to a molecule, generally an antibody orfusion proteins which blocks or reduces the effects of a specificmolecule on immunity. Generally in the present application this willrefer to anti-human VISTA antagonist antibodies and antibody fragmentswhich block or reduce the effects of human VISTA on immunity,particularly VISTA's suppressive effects on T cell immunity (CD4⁺ and/orCD8+ T cell immunity), the expression of proinflammatory cytokines andVISTA's effects of the expression of specific chemokines andchemoattractants.

“Antibody”, as used herein, refers broadly to an “antigen-bindingportion” of an antibody (also used interchangeably with “antibodyportion,” “antigen-binding fragment,” “antibody fragment”), as well aswhole antibody molecules. The term “antigen-binding portion”, as usedherein, refers to one or more fragments of an antibody that retain theability to specifically bind to an antigen (e.g., VISTA or specificportions thereof)). The term “antibody” as referred to herein includeswhole polyclonal and monoclonal antibodies and any antigen-bindingfragment (i.e., “antigen-binding portion”) or single chains thereof aswell as bispecific and multispecific antibodies, e.g., those that bindto multiple antigens or multiple antigen epitopes. An “antibody” refersto a glycoprotein comprising at least two heavy (H) chains and two light(L) chains interconnected by disulfide bonds, or an antigen-bindingportion thereof. Each heavy chain is comprised of at least one heavychain variable region (abbreviated herein as VH) and a heavy chainconstant region. The heavy chain constant region is comprised of threedomains, C_(H1), Cm and Cm—Each light chain is comprised of at least onelight chain variable region (abbreviated herein as V_(L)) and a lightchain constant region. The light chain constant region is comprised ofone domain, CL—The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FRs). Each VH and VL is composed of three CDRsand four FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies maymediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (C1q) of the classical complement system. Moregenerally, the term “antibody” is intended to include any polypeptidechain-containing molecular structure with a specific shape that fits toand recognizes an epitope, where one or more non-covalent bindinginteractions stabilize the complex between the molecular structure andthe epitope. The archetypal antibody molecule is the immunoglobulin, andall types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from allsources, e.g. human, rodent, rabbit, cow, sheep, pig, dog, othermammals, chicken, other avians, etc., are considered to be “antibodies.”

The antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Non-limiting examples ofantigen-binding fragments encompassed within the term “antigen-bindingportion” of an antibody include (a) a Fab fragment, a monovalentfragment consisting of the V_(I), V_(H), C_(I) and C_(H1) domains; (b) aF(ab′), fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (c) a Fd fragmentconsisting of the V_(H) and C_(H1) domains; (d) a Fv fragment consistingof the VL and VH domains of a single arm of an antibody; (e) a dAbfragment (Ward, et al. (1989) Nature 341: 544-546), which consists of aV_(H) domain; and (f) an isolated complementarily determining region(CDR). Furthermore, although the two domains of the Fv fragment, V_(L)and V_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(H) and V_(L) regions pair toform monovalent molecules (known as single chain Fv (scFv). See e.g.,Bird, et al. (1988) Science 242: 423-426; Huston, et al. (1988) ProcNatl. Acad. Sci. USA 85: 5879-5883; and Osbourn, et al. (1998) Nat.Biotechnol. 16: 778. Single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Any V_(H) and V_(L) sequences of specific scFv can be linked to humanimmunoglobulin constant region cDNA or genomic sequences, in order togenerate expression vectors encoding complete IgG molecules or otherisotypes. VH and V_(L) can also be used in the generation of Fab, Fv, orother fragments of immunoglobulins using either protein chemistry orrecombinant DNA technology. Other forms of single chain antibodies, suchas diabodies are also encompassed. Diabodies are bivalent, bispecificantibodies in which V_(H) and V_(L) domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen-binding sites. See e.g. Holliger, et al. (1993) Proc Natl.Acad. Sci. USA 90: 6444-6448; Poljak, et al. (1994) Structure 2:1121-1123. Still further, an antibody or antigen-binding portion thereof(antigen-binding fragment, antibody fragment, antibody portion) may bepart of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of immunoadhesion moleculesinclude use of the streptavidin core region to make a tetrameric scFvmolecule (Kipriyanov, et al. (1995) Hum. Antibodies Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminalpolyhistidine tag to make bivalent and biotinylated scFv molecules.Kipriyanov, et al. (1994) Mol. Immunol. 31: 1047-1058. Antibodyportions, such as Fab and F(ab′)₂ fragments, can be prepared from wholeantibodies using conventional techniques, such as papain or pepsindigestion, respectively, of whole antibodies. Moreover, antibodies,antibody portions and immunoadhesion molecules can be obtained usingstandard recombinant DNA techniques, as described herein. Antibodies maybe polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, ormodified forms thereof, e.g., humanized, chimeric, bispecific ormultispecific antibodies.

“Antibody recognizing an antigen” and “an antibody specific for anantigen” is used interchangeably herein with the term “an antibody whichbinds specifically to an antigen” and refers to an immunoglobulin orfragment thereof that specifically binds an antigen.

“Antigen,” as used herein, refers broadly to a molecule or a portion ofa molecule capable of being bound by an antibody which is additionallycapable of inducing an animal to produce an antibody capable of bindingto an epitope of that antigen. An antigen may have one epitope, or havemore than one epitope. The specific reaction referred to hereinindicates that the antigen will react, in a highly selective manner,with its corresponding antibody and not with the multitude of otherantibodies which may be evoked by other antigens. In the case of adesired enhanced immune response to particular antigens of interest,antigens include, but are not limited to; infectious disease antigensfor which a protective immune response may be elicited are exemplary.

“Antigen presenting cell,” as used herein, refers broadly toprofessional antigen presenting cells (e.g., B lymphocytes, monocytes,dendritic cells, and Langerhans cells) as well as other antigenpresenting cells (e.g., keratinocytes, endothelial cells, astrocytes,fibroblasts, and oligodendrocytes).

“Antisense nucleic acid molecule,” as used herein, refers broadly to anucleotide sequence which is complementary to a “sense” nucleic ac*idencoding a protein (e.g., complementary to the coding strand of adouble-stranded cDNA molecule) complementary to an mRNA sequence orcomplementary to the coding strand of a gene. Accordingly, antisensenucleic acid molecules can hydrogen bond to sense nucleic acidmolecules.

“Apoptosis,” as used herein, refers broadly to programmed cell deathwhich can be characterized using techniques which are known in the art.Apoptotic cell death can be characterized by cell shrinkage, membraneblebbing, and chromatin condensation culminating in cell fragmentation.Cells undergoing apoptosis also display a characteristic pattern ofinternucleosomal DNA cleavage.

“Autoimmunity” or “autoimmune disease or condition,” as used herein,refers broadly to a disease or disorder arising from and directedagainst an individual's own tissues or a co-segregate or manifestationthereof or resulting condition therefrom, and includes. Hereinautoimmune conditions include inflammatory or allergic conditions, e.g.,chronic diseases characterized by a host immune reaction againstself-antigens potentially associated with tissue destruction such asrheumatoid arthritis.

“B cell receptor” (BCR),” as used herein, refers broadly to the complexbetween membrane Ig (mIg) and other transmembrane polypeptides (e.g.,IgA. and Ig) found on B cells. The signal transduction function of mIgis triggered by crosslinking of receptor molecules by oligomeric ormultimeric antigens. B cells can also be activated byanti-immunoglobulin antibodies. Upon BCR activation, numerous changesoccur in B cells, including tyrosine phosphorylation.

“Cancer,” as used herein, refers broadly to any neoplastic disease(whether invasive or metastatic) characterized by abnormal anduncontrolled cell division causing malignant growth or tumor (e.g.,unregulated cell growth.) The term “cancer” or “cancerous” as usedherein should be understood to encompass any neoplastic disease (whetherinvasive, non-invasive or metastatic) which is characterized by abnormaland uncontrolled cell division causing malignant growth or tumor,non-limiting examples of which are described herein. This includes anyphysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer are exemplified in theworking examples. Further cancers include but are not limited to,carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularexamples of such cancers include squamous cell cancer, lung cancer(including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer), pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastsleukemia; multiple myeloma and post-transplant lymphoproliferativedisorder (PTLD). Other cancers amenable for treatment by the presentinvention include, but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include colorectal, bladder,ovarian, melanoma, squamous cell cancer, lung cancer (includingsmall-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, and squamous carcinoma of the lung), cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer (includinggastrointestinal cancer), pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.Preferably, the cancer is selected from the group consisting ofcolorectal cancer, breast cancer, colorectal cancer, rectal cancer,non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), renal cellcancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissuesarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer,melanoma, ovarian cancer, mesothelioma, and multiple myeloma. In anexemplary embodiment the cancer is an early or advanced (includingmetastatic) bladder, ovarian or melanoma. In another embodiment thecancer is colorectal cancer. The cancerous conditions amenable fortreatment of the invention include cancers that express or do notexpress VISTA and further include non-metastatic or non-invasive as wellas invasive or metastatic cancers wherein VISTA expression by immune,stromal or diseased cells suppress antitumor responses and anti-invasiveimmune responses. The method of the present invention is particularlysuitable for the treatment of vascularized tumors. Cancers according tothe invention include cancers that express or do not express VISTA andfurther include non-metastatic or non-invasive as well as invasive ormetastatic cancers wherein VISTA expression by immune, stromal ordiseased cells suppress antitumor responses and anti-invasive immuneresponses, and those characterized by vascularized tumors.

“Chimeric antibody,” as used herein, refers broadly to an antibodymolecule in which the constant region, or a portion thereof, is altered,replaced or exchanged so that the antigen-binding site (variable region)is linked to a constant region of a different or altered class, effectorfunction and/or species, or an entirely different molecule which confersnew properties to the chimeric antibody, e.g., an enzyme, toxin,hormone, growth factor, drug, the variable region or a portion thereof,is altered, replaced or exchanged with a variable region having adifferent or altered antigen specificity.

“Coding region,” as used herein, refers broadly to regions of anucleotide sequence comprising codons which are translated into aminoacid residues, whereas the term “noncoding region” refers to regions ofa nucleotide sequence that are not translated into amino acids (e.g., 5′and 3′ untranslated regions).

“Conservatively modified variants,” as used herein, applies to bothamino acid and nucleic acid sequences, and with respect to particularnucleic acid sequences, refers broadly to conservatively modifiedvariants refers to those nucleic acids which encode identical oressentially identical amino acid sequences, or where the nucleic aciddoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given protein.“Silent variations” are one species of conservatively modified nucleicacid variations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) may be modifiedto yield a functionally identical molecule.

“Complementarity determining region,” “hypervariable region,” or “CDR,”as used herein, refers broadly to one or more of the hyper-variable orcomplementarily determining regions (CDRs) found in the variable regionsof light or heavy chains of an antibody. See Kabat, et al. (1987)Sequences of Proteins of Immunological Interest National Institutes ofHealth, Bethesda, Md. These expressions include the hypervariableregions as defined by Kabat, et al. (1983) Sequences of Proteins ofImmunological Interest, U. S. Dept. of Health and Human Services or thehypervariable loops in 3-dimensional structures of antibodies. Chothiaand Lesk (1987) J. Mol. Biol. 196: 901-917. The CDRs in each chain areheld in close proximity by framework regions and, with the CDRs from theother chain, contribute to the formation of the antigen-binding site.Within the CDRs there are select amino acids that have been described asthe selectivity determining regions (SDRs) which represent the criticalcontact residues used by the CDR in the antibody-antigen interaction.(Kashmiri Methods 36: 25-34(2005)).

“Control amount,” as used herein, refers broadly to a marker can be anyamount or a range of amounts to be compared against a test amount of amarker. For example, a control amount of a marker may be the amount of amarker in a patient with a particular disease or condition or a personwithout such a disease or condition. A control amount can be either inabsolute amount (e.g., microgram/ml) or a relative amount (e.g.,relative intensity of signals).

“Costimulatory receptor,” as used herein, refers broadly to receptorswhich transmit a costimulatory signal to an immune cell, e.g., CD28 orICOS. As used herein, the term “inhibitory receptors” includes receptorswhich transmit a negative signal to an immune cell, e.g., a T cell or anNK cell.

“Costimulate,” as used herein, refers broadly to the ability of acostimulatory molecule to provide a second, non-activating,receptor-mediated signal (a “costimulatory signal”) that inducesproliferation or effector function. For example, a costimulatory signalcan result in cytokine secretion (e.g., in a T cell that has received aT cell-receptor-mediated signal) Immune cells that have received a cellreceptor-mediated signal (e.g., via an activating receptor) may bereferred to herein as “activated immune cells.” With respect to T cells,transmission of a costimulatory signal to a T cell involves a signalingpathway that is not inhibited by cyclosporin A. In addition, acostimulatory signal can induce cytokine secretion (e.g., IL-2 and/orIL-10) in a T cell and/or can prevent the induction of unresponsivenessto antigen, the induction of anergy, or the induction of cell death inthe T cell.

“Costimulatory polypeptide” or “costimulatory molecule” herein refers toa polypeptide that, upon interaction with a cell-surface molecule on Tcells, modulates T cell responses.

“Costimulatory signaling” as used herein is the signaling activityresulting from the interaction between costimulatory polypeptides onantigen presenting cells and their receptors on T cells duringantigen-specific T cell responses. Without wishing to be limited by asingle hypothesis, the antigen-specific T cell response is believed tobe mediated by two signals: 1) engagement of the T cell Receptor (TCR)with antigenic peptide presented in the context of MHC (signal 1), and2) a second antigen-independent signal delivered by contact betweendifferent costimulatory receptor/ligand pairs (signal 2). Withoutwishing to be limited by a single hypothesis, this “second signal” iscritical in determining the type of T cell response (activation vsinhibition) as well as the strength and duration of that response, andis regulated by both positive and negative signals from costimulatorymolecules, such as the B7 family of proteins.

“B7” polypeptide herein means a member of the B7 family of proteins thatcostimulate T cells including, but not limited to B7-1, B7-2, B7-DC,B7-H5, B7-HI, B7-H2, B7-H3, B7-H4, B7-H6, B7-53 and biologically activefragments and/or variants thereof. Representative biologically activefragments include the extracellular domain or fragments of theextracellular domain that costimulate T cells.

“Cytoplasmic domain,” as used herein, refers broadly to the portion of aprotein which extends into the cytoplasm of a cell.

“Diagnostic,” as used herein, refers broadly to identifying the presenceor nature of a pathologic condition. Diagnostic methods differ in theirsensitivity and specificity. The “sensitivity” of a diagnostic assay isthe percentage of diseased individuals who test positive (percent of“true positives”). Diseased individuals not detected by the assay are“false negatives.” Subjects who are not diseased and who test negativein the assay are termed “true negatives.” The “specificity” of adiagnostic assay is 1 minus the false positive rate, where the “falsepositive” rate is defined as the proportion of those without the diseasewho test positive. While a particular diagnostic method may not providea definitive diagnosis of a condition, it suffices if the methodprovides a positive indication that aids in diagnosis.

“Diagnosing,” or “aiding in the diagnosis” as used herein refers broadlyto classifying a disease or a symptom, and/or determining the likelihoodthat an individual has a disease condition (e.g., based on absence orpresence of VISTA expression, and/or increased or decreased expressionby immune, stromal and/or putative diseased cells); determining aseverity of the disease, monitoring disease progression, forecasting anoutcome of a disease and/or prospects of recovery. The term “detecting”may also optionally encompass any of the foregoing. Diagnosis of adisease according to the present invention may, in some embodiments, beaffected by determining a level of a polynucleotide or a polypeptide ofthe present invention in a biological sample obtained from the subject,wherein the level determined can be correlated with predisposition to,or presence or absence of the disease. It should be noted that a“biological sample obtained from the subject” may also optionallycomprise a sample that has not been physically removed from the subject.

“Effective amount,” as used herein, refers broadly to the amount of acompound, antibody, antigen, or cells that, when administered to apatient for treating a disease, is sufficient to effect such treatmentfor the disease. The effective amount may be an amount effective forprophylaxis, and/or an amount effective for prevention. The effectiveamount may be an amount effective to reduce, an amount effective toprevent the incidence of signs/symptoms, to reduce the severity of theincidence of signs/symptoms, to eliminate the incidence ofsigns/symptoms, to slow the development of the incidence ofsigns/symptoms, to prevent the development of the incidence ofsigns/symptoms, and/or effect prophylaxis of the incidence ofsigns/symptoms. The “effective amount” may vary depending on the diseaseand its severity and the age, weight, medical history, susceptibility,and pre-existing conditions, of the patient to be treated. The term“effective amount” is synonymous with “therapeutically effective amount”for purposes of this invention.

“Extracellular domain” or “ECD” as used herein refers broadly to theportion of a protein that extends from the surface of a cell.

“Expression vector,” as used herein, refers broadly to any recombinantexpression system for the purpose of expressing a nucleic acid sequenceof the invention in vitro or in vivo, constitutively or inducibly, inany cell, including prokaryotic, yeast, fungal, plant, insect ormammalian cell. The term includes linear or circular expression systems.The term includes expression systems that remain episomal or integrateinto the host cell genome. The expression systems can have the abilityto self-replicate or not, i.e., drive only transient expression in acell. The term includes recombinant expression cassettes which containonly the minimum elements needed for transcription of the recombinantnucleic acid.

“Family,” as used herein, refers broadly to the polypeptide and nucleicacid molecules of the invention is intended to mean two or morepolypeptide or nucleic acid molecules having a common structural domainor motif and having sufficient amino acid or nucleotide sequencehomology as defined herein. Family members can be naturally ornon-naturally occurring and can be from either the same or differentspecies. For example, a family can contain a first polypeptide of humanorigin, as well as other, distinct polypeptides of human origin oralternatively, can contain homologues of non-human origin (e.g., monkeypolypeptides.) Members of a family may also have common functionalcharacteristics.

“Fc receptor” (FcRs) as used herein, refers broadly to cell surfacereceptors for the Fc portion of immunoglobulin molecules (Igs). Fcreceptors are found on many cells which participate in immune responses.Among the human FcRs that have been identified so far are those whichrecognize IgG (designated FcyR), IgE (FceRI), IgA (FcaR), andpolymerized IgM/A (FcεμR). FcRs are found in the following cell types:FceRI (mast cells), FceRII (many leukocytes), FcaR (neutrophils), andFcpR (glandular epithelium, hepatocytes). (Hogg Immunol. Today 9: 185-86(1988)). The widely studied FcyRs are central in cellular immunedefenses, and are responsible for stimulating the release of mediatorsof inflammation and hydrolytic enzymes involved in the pathogenesis ofautoimmune disease. (Unkeless, Annu. Rev. Immunol. 6: 251-87 (1988)).The FcyRs provide a crucial link between effector cells and thelymphocytes that secrete Ig, since the macrophage/monocyte,polymorphonuclear leukocyte, and natural killer (NK) cell FcyRs conferan element of specific recognition mediated by IgG. Human leukocyteshave at least three different types of FcyRs for IgG: hFcγRI (CD64)(found on monocytes/macrophages), hFcyRIIA or hFcyRIIB, (CD32 or CD32A)(found on monocytes, neutrophils, eosinophils, platelets, possibly Bcells, and the K562 cell line) and FcγRIIIA (CD16A) or FcγRIIIB (CD16B)(found on NK cells, neutrophils, eosinophils, and macrophages).

“Framework region” or “FR,” as used herein refers broadly to one or moreof the framework regions within the variable regions of the light andheavy chains of an antibody. See Kabat, et al. Sequences of Proteins ofImmunological Interest National Institutes of Health, Bethesda, Md.(1987). These expressions include those amino acid sequence regionsinterposed between the CDRs within the variable regions of the light andheavy chains of an antibody.

“Graft versus Host Disease” (GVHD): as used herein refers to a commoncomplication of allogeneic bone marrow transplantation or hematopoieticstem cells transplantation in which functional immune cells in thetransplanted marrow recognize the recipient as “foreign” and produce animmune response to the host tissue. According to the 1959 BillinghamCriteria, there are three criteria must be met in order for GVHD tooccur: 1) Administration of an immunocompetent graft, with viable andfunctional immune cells; 2) the recipient is immunologicallyhistoincompatible; 3) The recipient is immunocompromised and thereforecannot destroy or inactivate the transplanted cells. Clinically,graft-versus-host-disease is divided into acute and chronic forms. Theacute or fulminant form of the disease (aGVHD) is normally observedwithin the first 100 days post-transplant, and is a major challenge tothe effectiveness of transplants owing to the associated morbidity andmortality. The chronic form of graft-versus-host-disease (cGVHD)normally occurs after 100 days. The appearance of moderate to severecases of cGVHD adversely influences long-term survival. After bonemarrow transplantation, T cells present in the graft, either ascontaminants or intentionally introduced into the host, attack thetissues of the transplant recipient after perceiving host tissues asantigenically foreign. The T cells produce an excess of cytokines,including TNF alpha and interferon-gamma (IFNγ). A wide range of hostantigens can initiate graft-versus-host-disease, among them the humanleukocyte antigens (HLAs). However, graft-versus-host disease can occureven when HLA-identical siblings are the donors. Classically, acutegraft-versus-host-disease is characterized by selective damage to theliver, skin and mucosa, and the gastrointestinal tract. Additionalstudies show that that graft-versus-host-disease targets organsincluding the immune system (such as the bone marrow and the thymus)itself, and the lungs in the form of idiopathic pneumonitis. Chronicgraft-versus-host-disease also attacks the above organs, but over itslong-term course can also cause damage to the connective tissue andexocrine glands.

“Heterologous,” as used herein, refers broadly to portions of a nucleicacid indicates that the nucleic acid comprises two or more subsequencesthat are not found in the same relationship to each other in nature. Forinstance, the nucleic acid is typically recombinantly produced, havingtwo or more sequences from unrelated genes arranged to make a newfunctional nucleic acid (e.g., a promoter from one source and a codingregion from another source.) Similarly, a heterologous protein indicatesthat the protein comprises two or more subsequences that are not foundin the same relationship to each other in nature (e.g., a fusionprotein).

“High affinity,” as used herein, refers broadly to an antibody or fusionprotein having a KD of at least 10−6 M, more preferably 10−7 M, evenmore preferably at least 10−8 M and even more preferably at least 10−9M, 10−10 M, 10−11 M, or 10−12 M for a target antigen or receptor. “Highaffinity” for an IgG antibody or fusion protein herein refers to anantibody having a KD of 10−6 M or less, more preferably 10−7 M or less,preferably 10−8 M or less, more preferably 10−9 M or less and even morepreferably 10−10 M, 10−11 M, or 10−12 M or less for a target antigen orreceptor. With particular respect to antibodies, “high affinity” bindingcan vary for different antibody isotypes. For example, “high affinity”binding for an IgM isotype refers to an antibody having a KD of 10−7 Mor less, more preferably 10−8 M or less.

“Homology,” as used herein, refers broadly to a degree of similaritybetween a nucleic acid sequence and a reference nucleic acid sequence orbetween a polypeptide sequence and a reference polypeptide sequence.Homology may be partial or complete. Complete homology indicates thatthe nucleic acid or amino acid sequences are identical. A partiallyhomologous nucleic acid or amino acid sequence is one that is notidentical to the reference nucleic acid or amino acid sequence. Thedegree of homology can be determined by sequence comparison, for exampleusing BlastP software of the National Center of BiotechnologyInformation (NCBI) using default parameters. The term “sequenceidentity” may be used interchangeably with “homology.”

“Host cell,” as used herein, refers broadly to refer to a cell intowhich a nucleic acid molecule of the invention, such as a recombinantexpression vector of the invention, has been introduced. Host cells maybe prokaryotic cells (e.g., E. coli), or eukaryotic cells such as yeast,insect (e.g., SF9), amphibian, or mammalian cells such as CHO, HeLa,HEK-293, e.g., cultured cells, explants, and cells in vivo. The terms“host cell” and “recombinant host cell” are used interchangeably herein.It should be understood that such terms refer not only to the particularsubject cell but to the progeny or potential progeny of such a cell.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, progeny may not, in fact,be identical to the parent cell, but are still included within the scopeof the term as used herein.

“Human monoclonal antibody” refers to antibodies displaying a singlebinding specificity which have variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. In one embodiment, the human monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from atransgenic nonhuman animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell. This includes fully human monoclonalantibodies and conjugates and variants thereof, e.g., which are bound toeffector agents such as therapeutics or diagnostic agents.

“Humanized antibody,” as used herein, refers broadly to includeantibodies made by a non-human cell having variable and constant regionswhich have been altered to more closely resemble antibodies that wouldbe made by a human cell. For example, by altering the non-human antibodyamino acid sequence to incorporate amino acids found in human germlineimmunoglobulin sequences. The humanized antibodies of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs. The term “humanized antibody”, as used herein, also includesantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences.

“Hybridization,” as used herein, refers broadly to the physicalinteraction of complementary (including partially complementary)polynucleotide strands by the formation of hydrogen bonds betweencomplementary nucleotides when the strands are arranged antiparallel toeach other.

“IgV domain” and “IgC domain” as used herein, refer broadly to Igsuperfamily member domains. These domains correspond to structural unitsthat have distinct folding patterns called Ig folds. Ig folds arecomprised of a sandwich of two 0 sheets, each consisting of antiparallelP strands of 5-10 amino acids with a conserved disulfide bond betweenthe two sheets in most, but not all, domains. IgC domains of Ig, TCR,and MHC molecules share the same types of sequence patterns and arecalled the CI set within the Ig superfamily. Other IgC domains fallwithin other sets. IgV domains also share sequence patter and are calledV set domains. IgV domains are longer than C-domains and form anadditional pair of β strands.

“Immune cell,” as used herein, refers broadly to cells that are ofhematopoietic origin and that play a role in the immune response. Immunecells include but are not limited to lymphocytes, such as B cells and Tcells; natural killer cells; dendritic cells, and myeloid cells, such asmonocytes, macrophages, eosinophils, mast cells, basophils, andgranulocytes.

“Immunoassay,” as used herein, refers broadly to an assay that uses anantibody to specifically bind an antigen. The immunoassay may becharacterized by the use of specific binding properties of a particularantibody to isolate, target, and/or quantify the antigen.

“Immune related disease (or disorder or condition)” as used hereinshould be understood to encompass any disease disorder or conditionselected from the group including but not limited to autoimmunediseases, inflammatory disorders and immune disorders associated withgraft transplantation rejection, such as acute and chronic rejection oforgan transplantation, allogenic stem cell transplantation, autologousstem cell transplantation, bone marrow transplantation, and graft versushost disease.

“Immune response,” as used herein, refers broadly to T cell-mediatedand/or B cell-mediated immune responses that are influenced bymodulation of T cell costimulation. Exemplary immune responses include Bcell responses (e.g., antibody production) T cell responses (e.g.,cytokine production, and cellular cytotoxicity) and activation ofcytokine responsive cells, e.g., macrophages. As used herein, the term“downmodulation” with reference to the immune response includes adiminution in any one or more immune responses, while the term“upmodulation” with reference to the immune response includes anincrease in any one or more immune responses. It will be understood thatupmodulation of one type of immune response may lead to a correspondingdownmodulation in another type of immune response. For example,upmodulation of the production of certain cytokines (e.g., IL-10) canlead to downmodulation of cellular immune responses.

“Immunologic”, “immunological” or “immune” response herein refer to thedevelopment of a humoral (antibody mediated) and/or a cellular (mediatedby antigen-specific T cells or their secretion products) responsedirected against a peptide in a recipient patient. Such a response canbe an active response induced by administration of immunogen or apassive response induced by administration of antibody or primedT-cells. Without wishing to be limited by a single hypothesis, acellular immune response is elicited by the presentation of polypeptideepitopes in association with Class II or Class I MHC molecules toactivate antigen-specific CD4 T helper cells and/or CD8, cytotoxic Tcells, respectively. The response may also involve activation ofmonocytes, macrophages, NK cells, basophils, dendritic cells,astrocytes, microglia cells, eosinophils, activation or recruitment ofneutrophils or other components of innate immunity. The presence of acell-mediated immunological response can be determined by proliferationassays (CD4 T cells) or CTL (cytotoxic T lymphocyte) assays. Therelative contributions of humoral and cellular responses to theprotective or therapeutic effect of an immunogen can be distinguished byseparately isolating antibodies and T cells from an immunized syngeneicanimal and measuring protective or therapeutic effect in a secondsubject.

“Immunogenic agent” or “immunogen” is a moiety capable of inducing animmunological response against itself on administration to a mammal,optionally in conjunction with an adjuvant.

“Inflammatory disorders”, “inflammatory conditions” and/or“inflammation”, used interchangeably herein, refers broadly to chronicor acute inflammatory diseases, and expressly includes inflammatoryautoimmune diseases and inflammatory allergic conditions. Theseconditions include by way of example inflammatory abnormalitiescharacterized by dysregulated immune response to harmful stimuli, suchas pathogens, damaged cells, or irritants. Inflammatory disordersunderlie a vast variety of human diseases. Non-immune diseases withetiological origins in inflammatory processes include cancer,atherosclerosis, and ischemic heart disease. Examples of disordersassociated with inflammation include: Chronic prostatitis,Glomerulonephritis, Hypersensitivities, Pelvic inflammatory disease,Reperfusion injury, Sarcoidosis, Vasculitis, Interstitial cystitis,normocomplementemic urticarial vasculitis, pericarditis, myositis,anti-synthetase syndrome, scleritis, macrophage activation syndrome,Behçet's Syndrome, PAPA Syndrome, Blau's Syndrome, gout, adult andjuvenile Still's disease, cryropyrinopathy, Muckle-Wells syndrome,familial cold-induced auto-inflammatory syndrome, neonatal onsetmultisystemic inflammatory disease, familial Mediterranean fever,chronic infantile neurologic, cutaneous and articular syndrome, systemicjuvenile idiopathic arthritis, Hyper IgD syndrome, Schnitzler'ssyndrome, TNF receptor-associated periodic syndrome (TRAPSP),gingivitis, periodontitis, hepatitis, cirrhosis, pancreatitis,myocarditis, vasculitis, gastritis, gout, gouty arthritis, andinflammatory skin disorders, selected from the group consisting ofpsoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne.

“Inhibitory signal,” as used herein, refers broadly to a signaltransmitted via an inhibitory receptor molecule on an immune cell. Asignal antagonizes a signal via an activating receptor (e.g., via a TCR,CD3, BCR, or Fc molecule) and can result, e.g., in inhibition of: secondmessenger generation; proliferation; or effector function in the immunecell, e.g., reduced phagocytosis, antibody production, or cellularcytotoxicity, or the failure of the immune cell to produce mediators(e.g., cytokines (e.g., IL-2) and/or mediators of allergic responses);or the development of anergy.

“Isolated,” as used herein, refers broadly to material removed from itsoriginal environment in which it naturally occurs, and thus is alteredby the hand of man from its natural environment and includes“recombinant” polypeptides. Isolated material may be, for example,exogenous nucleic acid included in a vector system, exogenous nucleicacid contained within a host cell, or any material which has beenremoved from its original environment and thus altered by the hand ofman (e.g., “isolated antibody”). For example, “isolated” or “purified,”as used herein, refers broadly to a protein, DNA, antibody, RNA, orbiologically active portion thereof, that is substantially free ofcellular material or other contaminating proteins from the cell ortissue source from which the biological substance is derived, orsubstantially free from chemical precursors or other chemicals whenchemically synthesized. As used herein the term “isolated” refers to acompound of interest (for example a polynucleotide or a polypeptide)that is in an environment different from that in which the compoundnaturally occurs e.g., separated from its natural milieu such as byconcentrating a peptide to a concentration at which it is not found innature. “Isolated” includes compounds that are within samples that aresubstantially enriched for the compound of interest and/or in which thecompound of interest is partially or substantially purified.

“Isolated antibody”, as used herein, is intended to refer to an antibodythat is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds VISTA) is substantially free of antibodies that specifically bindantigens other than VISTA). Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

“Isotype” herein refers to the antibody class (e.g., IgM or IgG1) thatis encoded by the heavy chain constant region genes.

“K-assoc” or “Ka”, as used herein, refers broadly to the associationrate of a particular antibody-antigen interaction, whereas the term“Kdiss” or “Kd,” as used herein, refers to the dissociation rate of aparticular antibody-antigen interaction.

The term “K_(D)”, as used herein, is intended to refer to thedissociation constant, which is obtained from the ratio of Kd to Ka(i.e., Kd/Ka) and is expressed as a molar concentration (M). K_(D)values for antibodies can be determined using methods well establishedin the art such as plasmon resonance (BIAcore®), ELISA and KINEXA. Apreferred method for determining the K_(D) of an antibody is by usingsurface Plasmon resonance, preferably using a biosensor system such as aBIAcore® system or by ELISA. Typically these methods are effected at 25°or 37° C. Antibodies for therapeutic usage generally will possess aK_(D) when determined by surface Plasmon resonance of 50 nM or less ormore typically 1 nM or less at 25° or 37° C.

“Label” or a “detectable moiety” as used herein, refers broadly to acomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, chemical, or other physical means.

“Low stringency,” “medium stringency,” “high stringency,” or “very highstringency conditions,” as used herein, refers broadly to conditions fornucleic acid hybridization and washing. Guidance for performinghybridization reactions can be found in Ausubel, et al., Short Protocolsin Molecular Biology (5th Ed.) John Wiley & Sons, NY (2002). Exemplaryspecific hybridization conditions include but are not limited to: (1)low stringency hybridization conditions in 6× sodium chloride/sodiumcitrate (SSC) at about 45′ C, followed by two washes in 0.2×SSC, 0.1%SDS at least at 50° C. (the temperature of the washes can be increasedto 55° C. for low stringency conditions); (2) medium stringencyhybridization conditions in 6×SSC at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 60° C.; (3) high stringencyhybridization conditions in 6×SSC at about 45° C. followed by one ormore washes in 0.2×SSC, 0.1% SDS at 65° C.; and (4) very high stringencyhybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C.,followed by one or more washes at 0.2×SSC, and 1% SDS at 65° C.

“Lupus”, as used herein, is intended to include all types of lupus.There are 4 types of lupus which are discussed below. “Lupus-likecondition”, as used herein, is intended to include inflammatoryconditions with symptoms similar to lupus such as kidney inflammation,increased proteinuria, and splenomegaly. “Systemic Lupus Erythematosus”or (“SLE”) the most common form of lupus which can be mild or severe andcan affect major organ systems. This is the condition most peopleassociate with “lupus”. It is an autoimmune condition of unknown causethat may result in inflammation of the kidneys—called lupusnephritis—which can affect the body's ability to filter waste from theblood, and or if severe may result in kidney damage requiring dialysisor kidney transplant. Also SLE may result in an increase in bloodpressure in the lungs-called pulmonary hypertension—can cause difficultybreathing. Further SLE may cause Inflammation of the nervous system andbrain which can cause memory problems, confusion, headaches, andstrokes. Further SLE may result in inflammation in the brain's bloodvessels which can cause high fevers, seizures, and behavioral changes.Also SLE may result in hardening of the arteries or coronary arterydisease—the buildup of deposits on coronary artery walls—can lead to aheart attack. “Skin Lupus” herein refers to lupus conditions that onlyaffect the skin. There are three types of lupus that affect the skinchronic cutaneous lupus erythematosus (CCLE) (also known as DiscoidLupus Erythematosus [DLE]), subacute cutaneous lupus erythematosus(SCLE), and tumid lupus. Cutaneous Lupus Erythematosus or Discoid LupusErythematosus can cause many types of rashes and lesions (sores), themost common—called discoid rash—is raised, scaly and red, but not itchy.Areas of rash appear like disks, or circles. Another common example ofcutaneous lupus is a rash over the cheeks and across the bridge of thenose, known as the butterfly rash. Other rashes or sores may appear onthe face, neck, or scalp (areas of the skin that are exposed to sunlightor fluorescent light), or in the mouth, nose, or vagina. Hair loss andchanges in the pigment, or color, of the skin are also symptoms ofcutaneous lupus. Approximately 10 percent of people who have cutaneouslupus will develop systemic lupus. However, it is likely that thesepeople already had systemic lupus, with the skin rash as their mainsymptom. “Drug-induced Lupus Erythematosus” is a condition caused bycertain drugs which can cause lupus-like symptoms in people who do nothave SLE. Generally, this form of lupus is temporary and usuallysubsides within months of the time that the medication is stopped.Medications known to induce lupus-like symptoms include the bloodpressure medications hydralazine and methyldopa, a heart medicationcalled procainamide, and a drug called D-penicillamine, which is used incases of metal poisoning. Other causes of drug-induced lupus includeminocycline (used to treat acne), Isoniazid—a treatment for tuberculosisand anti-TNF (used to treat rheumatoid arthritis). The symptoms ofdrug-induced lupus are similar to those of systemic lupus, howeverunlike SLE but it rarely affects major organs. Neonatal lupus is not atrue form of lupus. It is a rare condition that affects infants of womenwho have lupus and is caused by antibodies from the mother acting uponthe infant in the womb. At birth, the infant may have a skin rash, liverproblems, or low blood cell counts but these symptoms generallydisappear completely after several months with no lasting effects. Someinfants with neonatal lupus can also have a serious heart defect.

“Mammal,” as used herein, refers broadly to any and all warm-bloodedvertebrate animals of the class Mammalia, including humans,characterized by a covering of hair on the skin and, in the female,milk-producing mammary glands for nourishing the young. Examples ofmammals include but are not limited to alpacas, armadillos, capybaras,cats, camels, chimpanzees, chinchillas, cattle, dogs, goats, gorillas,hamsters, horses, humans, lemurs, llamas, mice, non-human primates,pigs, rats, sheep, shrews, squirrels, tapirs, and voles. Mammals includebut are not limited to bovine, canine, equine, feline, murine, ovine,porcine, primate, and rodent species. Mammal also includes any and allthose listed on the Mammal Species of the World maintained by theNational Museum of Natural History, Smithsonian Institution inWashington D. C.

“Multispecific antibody” refers to an antibody with 2 or more antigenbinding regions. This includes bispecific antibodies. These antigenbinding regions may bind to different antigens or to different epitopesof the same antigen.

“Naturally-occurring nucleic acid molecule,” as used herein, refersbroadly refers to an RNA or DNA molecule having a nucleotide sequencethat occurs in nature (e.g., encodes a natural protein).

“Nucleic acid” or “nucleic acid sequence,” as used herein, refersbroadly to a deoxy-ribonucleotide or ribonucleotide oligonucleotide ineither single- or double-stranded form. The term encompasses nucleicacids, i.e., oligonucleotides, containing known analogs of naturalnucleotides. The term also encompasses nucleic-acid-like structures withsynthetic backbones. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions) andcomplementary sequences, as well as the sequence explicitly indicated.The term nucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

“Operatively linked”, as used herein, refers broadly to when two DNAfragments are joined such that the amino acid sequences encoded by thetwo DNA fragments remain in-frame.

“Paratope,” as used herein, refers broadly to the part of an antibodywhich recognizes an antigen (e.g., the antigen-binding site of anantibody.) Paratopes may be a small region (e.g., 15-22 amino acids) ofthe antibody's Fv region and may contain parts of the antibody's heavyand light chains. See Goldsby, et al. Antigens (Chapter 3) Immunology(5th Ed.) New York: W. H. Freeman and Company, pages 57-75.

“Patient,” or “subject” or “recipient”, “individual”, or “treatedindividual” are used interchangeably herein, and refers broadly to anyanimal that is in need of treatment either to alleviate a disease stateor to prevent the occurrence or reoccurrence of a disease state. Also,“Patient” as used herein, refers broadly to any animal that has riskfactors, a history of disease, susceptibility, symptoms, and signs, waspreviously diagnosed, is at risk for, or is a member of a patientpopulation for a disease. The patient may be a clinical patient such asa human or a veterinary patient such as a companion, domesticated,livestock, exotic, or zoo animal.

“Polypeptide,” “peptide” and “protein,” are used interchangeably andrefer broadly to a polymer of amino acid residues s of any length,regardless of modification (e.g., phosphorylation or glycosylation). Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer. Polypeptidescan be modified, e.g., by the addition of carbohydrate residues to formglycoproteins. The terms “polypeptide,” “peptide” and “protein”expressly include glycoproteins, as well as non-glycoproteins.

“Promoter,” as used herein, refers broadly to an array of nucleic acidsequences that direct transcription of a nucleic acid. As used herein, apromoter includes necessary nucleic acid sequences near the start siteof transcription, such as, in the case of a polymerase II type promoter,a TATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation.

“Prophylactically effective amount,” as used herein, refers broadly tothe amount of a compound that, when administered to a patient forprophylaxis of a disease or prevention of the reoccurrence of a disease,is sufficient to effect such prophylaxis for the disease orreoccurrence. The prophylactically effective amount may be an amounteffective to prevent the incidence of signs and/or symptoms. The“prophylactically effective amount” may vary depending on the diseaseand its severity and the age, weight, medical history, predisposition toconditions, preexisting conditions, of the patient to be treated.

“Prophylactic vaccine” and/or “Prophylactic vaccination” refers to avaccine used to prevent a disease or symptoms associated with a diseasesuch as cancer or an infectious condition.

“Prophylaxis,” as used herein, refers broadly to a course of therapywhere signs and/or symptoms are not present in the patient, are inremission, or were previously present in a patient. Prophylaxis includespreventing disease occurring subsequent to treatment of a disease in apatient. Further, prevention includes treating patients who maypotentially develop the disease, especially patients who are susceptibleto the disease (e.g., members of a patent population, those with riskfactors, or at risk for developing the disease).

“Recombinant” as used herein, refers broadly with reference to aproduct, e.g., to a cell, or nucleic acid, protein, or vector, indicatesthat the cell, nucleic acid, protein or vector, has been modified by theintroduction of a heterologous nucleic acid or protein or the alterationof a native nucleic acid or protein, or that the cell is derived from acell so modified. Thus, for example, recombinant cells express genesthat are not found within the native (non-recombinant) form of the cellor express native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(I) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

“Signal sequence” or “signal peptide,” as used herein, refers broadly toa peptide containing about 15 or more amino acids which occurs at theN-terminus of secretory and membrane bound polypeptides and whichcontains a large number of hydrophobic amino acid residues. For example,a signal sequence contains at least about 10-30 amino acid residues,preferably about 15-25 amino acid residues, more preferably about 18-20amino acid residues, and even more preferably about 19 amino acidresidues, and has at least about 35-65%, preferably about 38-50%, andmore preferably about 40-45% hydrophobic amino acid residues (e.g.,Valine, Leucine, Isoleucine or Phenylalanine). A “signal sequence,” alsoreferred to in the art as a “signal peptide,” serves to direct apolypeptide containing such a sequence to a lipid bilayer, and iscleaved in secreted.

“Specifically (or selectively) binds” to an antibody or “specifically(or selectively) immunoreactive with,” or “specifically interacts orbinds,” as used herein, refers broadly to a protein or peptide (or otherepitope), refers, in some embodiments, to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologies. For example, underdesignated immunoassay conditions, the specified antibodies bind to aparticular protein at least two times greater than the background(non-specific signal) and do not substantially bind in a significantamount to other proteins present in the sample. Typically a specific orselective reaction will be at least twice background signal or noise andmore typically more than about 10 to 100 times background.

“Specifically hybridizable” and “complementary” as used herein, referbroadly to a nucleic acid can form hydrogen bond(s) with another nucleicacid sequence by either traditional Watson-Crick or othernon-traditional types. The binding free energy for a nucleic acidmolecule with its complementary sequence is sufficient to allow therelevant function of the nucleic acid to proceed, e.g., RNAi activity.Determination of binding free energies for nucleic acid molecules iswell known in the art. (See, e.g., Turner, et al. CSH Symp. Quant. Biol.LII: 123-33 (1987); Frier, et al. PNAS 83: 9373-77 1986); Turner, et al.J. Am. Chem. Soc. 109:3783-85 (1987)). A percent complementarityindicates the percentage of contiguous residues in a nucleic acidmolecule that can form hydrogen bonds (e.g., Watson-Crick base pairing)with a second nucleic acid sequence (e.g., about at least 5, 6, 7, 8, 9,10 out of 10 being about at least 50%, 60%, 70%, 80%, 90%, and 100%complementary, inclusive). “Perfectly complementary” or 100%complementarity refers broadly all of the contiguous residues of anucleic acid sequence hydrogen bonding with the same number ofcontiguous residues in a second nucleic acid sequence.

“Substantial complementarity” refers to polynucleotide strandsexhibiting about at least 90% complementarity, excluding regions of thepolynucleotide strands, such as overhangs, that are selected so as to benoncomplementary. Specific binding requires a sufficient degree ofcomplementarity to avoid non-specific binding of the oligomeric compoundto non-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, or in the case of in vitro assays,under conditions in which the assays are performed. The non-targetsequences typically may differ by at least 5 nucleotides.

“Signs” of disease, as used herein, refers broadly to any abnormalityindicative of disease, discoverable on examination of the patient; anobjective indication of disease, in contrast to a symptom, which is asubjective indication of disease.

“Solid support,” “support,” and “substrate,” as used herein, refersbroadly to any material that provides a solid or semi-solid structurewith which another material can be attached including but not limited tosmooth supports (e.g., metal, glass, plastic, silicon, and ceramicsurfaces) as well as textured and porous materials.

“Soluble ectodomain (ECD)” or “ectodomain” or “soluble VISTAprotein(s)/molecule(s)” of VISTA as used herein meansnon-cell-surface-bound VISTA molecules or any portion thereof,including, but not limited to: VISTA fusion proteins or VISTA ECD-Igfusion proteins, wherein the extracellular domain of VISTA or fragmentthereof is fused to an immunoglobulin (Ig) moiety rendering the fusionmolecule soluble, or fragments and derivatives thereof, proteins withthe extracellular domain of VISTA fused or joined with a portion of abiologically active or chemically active protein such as thepapillomavirus E7 gene product, melanoma-associated antigen p97 or HIVenv protein, or fragments and derivatives thereof; hybrid (chimeric)fusion proteins such as VISTA-Ig, or fragments and derivatives thereof.Such fusion proteins are described in greater detail below.

“Soluble VISTA protein(s)/molecule(s)” herein also include VISTAmolecules with the transmembrane domain removed to render the proteinsoluble, or fragments and derivatives thereof; fragments, portions orderivatives thereof, and soluble VISTA mutant molecules. The solubleVISTA molecules used in the methods according to at least someembodiments of the invention may or may not include a signal (leader)peptide sequence.

“Subject” or “patient” or “individual” in the context of therapy ordiagnosis herein includes any human or nonhuman animal. The term“nonhuman animal” includes all vertebrates, e.g., mammals andnon-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows,chickens, amphibians, reptiles, etc., i.e., anyone suitable to betreated according to the present invention include, but are not limitedto, avian and mammalian subjects, and are preferably mammalian. Anymammalian subject in need of being treated according to the presentinvention is suitable. Human subjects of both genders and at any stageof development (i.e., neonate, infant, juvenile, adolescent, and adult)can be treated according to the present invention. The present inventionmay also be carried out on animal subjects, particularly mammaliansubjects such as mice, rats, dogs, cats, cattle, goats, sheep, andhorses for veterinary purposes, and for drug screening and drugdevelopment purposes. “Subjects” is used interchangeably with“individuals” and “patients.”

“Substantially free of chemical precursors or other chemicals,” as usedherein, refers broadly to preparations of VISTA protein in which theprotein is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of VISTA protein having less than about 30% (bydry weight) of chemical precursors or non-VISTA chemicals, morepreferably less than about 20% chemical precursors or non-VISTAchemicals, still more preferably less than about 10% chemical precursorsor non-VISTA chemicals, and most preferably less than about 5% chemicalprecursors or non-VISTA chemicals.

“Symptoms” of disease as used herein, refers broadly to any morbidphenomenon or departure from the normal in structure, function, orsensation, experienced by the patient and indicative of disease.

“T cell,” as used herein, refers broadly to CD4+ T cells and CD8+ Tcells. The term T cell also includes both T helper 1 type T cells and Thelper 2 type T cells.

“Therapy,” “therapeutic,” “treating,” or “treatment”, as used herein,refers broadly to treating a disease, arresting, or reducing thedevelopment of the disease or its clinical symptoms, and/or relievingthe disease, causing regression of the disease or its clinical symptoms.Therapy encompasses prophylaxis, treatment, remedy, reduction,alleviation, and/or providing relief from a disease, signs, and/orsymptoms of a disease. Therapy encompasses an alleviation of signsand/or symptoms in patients with ongoing disease signs and/or symptoms(e.g., inflammation, pain). Therapy also encompasses “prophylaxis”. Theterm “reduced”, for purpose of therapy, refers broadly to the clinicalsignificant reduction in signs and/or symptoms. Therapy includestreating relapses or recurrent signs and/or symptoms (e.g.,inflammation, pain). Therapy encompasses but is not limited toprecluding the appearance of signs and/or symptoms anytime as well asreducing existing signs and/or symptoms and eliminating existing signsand/or symptoms. Therapy includes treating chronic disease(“maintenance”) and acute disease. For example, treatment includestreating or preventing relapses or the recurrence of signs and/orsymptoms (e.g., inflammation, pain).

“Treg cell” (sometimes also referred to as suppressor T cells orinducible Treg cells or iTregs) as used herein refers to a subpopulationof T cells which modulate the immune system and maintain tolerance toself-antigens and can abrogate autoimmune diseases. Foxp3⁺ CD4⁺ CD25⁺regulatory T cells (Tregs) are critical in maintaining peripheraltolerance under normal conditions.

“Transmembrane domain,” as used herein, refers broadly to an amino acidsequence of about 15 amino acid residues in length which spans theplasma membrane. More preferably, a transmembrane domain includes aboutat least 20, 25, 30, 35, 40, or 45 amino acid residues and spans theplasma membrane. Transmembrane domains are rich in hydrophobic residues,and typically have an a-helical structure. In an embodiment, at least50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of atransmembrane domain are hydrophobic, e.g., leucines, isoleucines,tyrosines, or tryptophans. Transmembrane domains are described in, forexample, Zagotta, et al. Annu. Rev. Neurosci. 19:235-263 (1996).

“Transgenic animal,” as used herein, refers broadly to a non-humananimal, preferably a mammal, more preferably a mouse, in which one ormore of the cells of the animal includes a “transgene”. The term“transgene” refers to exogenous DNA which is integrated into the genomeof a cell from which a transgenic animal develops and which remains inthe genome of the mature animal, for example directing the expression ofan encoded gene product in one or more cell types or tissues of thetransgenic animal.

“Unresponsiveness,” as used herein, refers broadly to refractivity ofimmune cells to stimulation, e.g., and stimulation via an activatingreceptor or a cytokine. Unresponsiveness can occur, e.g., because ofexposure to immunosuppressants or high doses of antigen.

“Variable region” or “VR,” as used herein, refers broadly to the domainswithin each pair of light and heavy chains in an antibody that areinvolved directly in binding the antibody to the antigen. Each heavychain has at one end a variable domain (V_(H)) followed by a number ofconstant domains. Each light chain has a variable domain (V_(L)) at oneend and a constant domain at its other end; the constant domain of thelight chain is aligned with the first constant domain of the heavychain, and the light chain variable domain is aligned with the variabledomain of the heavy chain.

“Vector,” as used herein, refers broadly to a nucleic acid moleculecapable of transporting another nucleic acid molecule to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Vectors are referred to herein as “recombinant expression vectors” orsimply “expression vectors”. In general, expression vectors of utilityin recombinant DNA techniques are often in the form of plasmids. In thepresent specification, “plasmid” and “vector” may be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions. The techniques and procedures are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. See, e.g., Sambrook,et al. Molec. Cloning: Lab. Manual [3rd Ed] Cold Spring HarborLaboratory Press (2001). Standard techniques may be used for recombinantDNA, oligonucleotide synthesis, and tissue culture, and transformation(e.g., electroporation, lipofection). Enzymatic reactions andpurification techniques may be performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein.

Having defined certain terms and phrases used in the presentapplication, the anti-VISTA antibodies and antigen binding antibodyfragments and methods for the production and use thereof which areembraced by the invention are further described below.

The present invention relates to antibodies and antibody fragmentscomprising an antigen binding region that binds to a V-domain IgSuppressor of T cell Activation (VISTA). VISTA is a checkpoint regulatorthat negatively suppresses immune responses. See Wang et al., “VISTA, anovel mouse Ig superfamily ligand that negatively regulates T cellresponses,” J. Exp. Med., 208(3) 577-92 (2011). This protein isexpressed on normal human neutrophils, monocytes and T cells subsets. Inaddition, cynomolgus monkey cells express VISTA in a similar pattern tonormal human cells. VISTA is also expressed in the peripheral bloodcells e.g., of cancer patients.

The binding of an agonist anti-VISTA antibody or antibody fragmentaccording to the invention will agonize, elicit or mimic at least one ofthe effects of VISTA on immunity thereby promoting at least one of thesuppressive effects of VISTA on immunity, e.g., the suppression of Tcell immunity or the suppression of the expression of specificproinflammatory cytokines or its promoting effect on the expression ofcertain chemoattractants and chemokines.

Such antibody fragments include by way of example Fab, F(ab′)₂, and scFvantibody fragments. These antibody or antibody fragments can comprise anantibody constant region or fragment or variant thereof. Such antibodiesand antibody fragments include those which bind to VISTA proteinsexpressed on hematopoietic and other cells, for example, myeloid cellsand/or lymphocytes, monocytes, neutrophils, T cells, natural killer (NK)cells, natural killer T (NKT) cells, a tumor cell, and/or in the tumormicroenvironment (TME). The tumor microenvironment is the cellularenvironment of the tumor. It can include surrounding immune cells,fibroblasts, blood vessels, other cells, signaling molecules, and theextracellular matrix.

The subject application provides novel agonist anti-human VISTAantibodies including those comprising the same CDRS as any of theanti-human VISTA antibodies having the sequences shown in FIG. 4. Whileprior to the present invention a number of antagonist anti-human VISTAantibodies have been reported in the literature, no agonistic anti-humanVISTA antibodies or antibody fragments have been reported.

As disclosed in the experimental examples which follow 2 chimericanti-human VISTA antibodies were initially derived from an antagonisticmurine anti-human VISTA antibody (1E8 having sequences in FIG. 4) whichrespectively contain unmodified IgG2 human constant regions or IgG2constant regions wherein the cysteine residue at position 127 of thekappa chain was changed to a serine residue. As shown in the Examplesand the Figures referenced therein, both antibodies were found toagonize or mimic the suppressive effects of VISTA on immunity at leastbased on (i) their ability to decrease the expression of certainproinflammatory cytokines such as IL-2, IL-4, IL-6, IL-17, granulocytemacrophage colony stimulating factor (GM-CSF) and tumor necrosisfactor-alpha (TNF-α) as well as reducing the expression of certainchemokines or chemoattractants such as KC (keratinocyte derivedchemokine) or MIP-2 (Macrophage Inflammatory Protein-2); (ii) suppress Tcell activity in GVHD model; and to (iii) suppress CD3-driven T cellresponses.

Additionally after isolation of these 2 agonist antibodies another 10chimeric agonist anti-human VISTA antibodies containing human IgG2constant or Fc regions have been obtained. These antibodies were derivedfrom the antibodies referred to herein as GG8, VSTB95 (INX903), VSTB103(INX904), VSTB53 (INX905), VSTB92 (INX908), VSTB50 (INX900), VSTB56(INX901), VSTB63 (INX902), VSTB54 (INX906) and VSTB66 (INX907)(havingthe sequences in FIG. 4).

Particularly, these chimeric anti-human VISTA antibodies have thevariable sequences shown in FIG. 4 and human IgG2 constant regions. Asreported in Tables 1 and 2 infra these anti-human VISTA antibodies whenassessed by use of antibody binning were found to bind to 2 differentepitope groups designated Group 1 and Group 2. As noted in the FIG. 4the epitope corresponding to Group 2 includes residues in 2 differentpeptides present in human VISTA, i.e., NLTLLDSGL and VQTGKDAPSNC. Also,as described in the examples infra the epitopic specificity of otheragonist antibodies according to the invention has been determined byPepscans analysis.

As is indicated in the Tables 1 and 2 infra these 12 differentanti-human VISTA antibodies were found to be immunosuppressive in atleast one model of immunosuppression and many in severalimmunosuppression models. Particularly INX905, INX908, INX901, INX902and INX906 were shown to be immunosuppressive in 2 different assaysformats. While all of these antibodies were immunosuppressive and appearto elicit, promote or agonize the immunosuppressive effects of VISTA,INX901, INX902 and INX906 and INX908 appear to be the mostimmunosuppressive.

Also, other chimeric anti-human VISTA antibodies comprising human IgG2constant domains containing the variable sequences of other anti-VISTAantibodies shown in FIG. 4 are to be screened for theirimmunosuppressive properties and their ability to agonize or mimic theimmunosuppressive and other effects of human VISTA. Based on the resultsobtained to date this screening should identify other agonist anti-humanVISTA antibodies. Additionally agonist anti-human VISTA antibodiesaccording to the invention have been shown to be effective(immunosuppressive) in numerous autoimmune and inflammatory animaldisease models including arthritis, lupus or SLE, GVHD, inflammatorybowel disease (IBD) or colitis, chronic and acute infectious disease orhepatotoxicity and psoriasis animal models. Based thereon the subjectanti-human VISTA agonist antibodies should be well suited for use intherapeutic and prophylactic treatment of autoimmune, allergic andinflammatory conditions.

As noted chimeric IgG2 anti-human VISTA antibodies having the sequencesshown in FIG. 4 were shown to be immunosuppressive in at least one modelof immunosuppression. These antibodies moreover elicit theseimmunosuppressive effects in a specific immunomodulatory manner ratherthan by effecting the depletion of specific types of T cells or bydepleting T cells in general.

As further shown in the examples surprisingly chimeric IgG2 agonisticanti-human VISTA antibodies containing a mutation in the hinge regionelicited substantially the same suppressive effects on immunity, i.e.,the mutation within in IgG2 constant region appeared to elicit noenhancement in suppression under the tested experimental conditions.Rather both the IgG2A and IgG2 B forms and mixtures thereof elicited thesame immunosuppressive effects. Additionally, and also surprisingly,based on experiments disclosed in the examples it would appear that FcγRbinding may contribute to the agonist properties of the subjectanti-human VISTA antibodies. In particular it was found that theinclusion of silent IgG2 constant regions ablated the immunosuppressiveproperties of the subject agonist antibodies. Based on these results itis hypothesized that one or more FcγRs may affect the agonisticproperties of these antibodies and in particular it is hypothesized thatFcγRIIA (CD32 or CD32A) or FcγRIIB (CD32B) binding may be involved inthe agonist properties of the subject agonist antibodies.

Using these same methods it is expected that other agonist anti-humanVISTA IgG2 antibodies may be obtained, e.g., others derived fromanti-human VISTA antibodies having the sequences shown in FIG. 4. Asmentioned 12 agonist anti-human VISTA antibodies have been obtained todate including those having the sequences contained in FIG. 4. Based onthese results it is anticipated that other agonistic anti-human VISTAantibodies may be generated and shown to be immunosuppressive. Also itis anticipated that other agonistic anti-human VISTA antibodies may begenerated which bind to the same or overlapping epitope and/or competewith any of the antibodies containing the sequences shown in FIG. 4. Inexemplary embodiments these antibodies will bind to the epitopecorresponding to Group 1 or Group 2 antibodies or will compete forbinding to human VISTA with such antibodies.

Methods for identifying the specific epitope(s) bound by an antibody areknown in the art. In the working examples Applicants disclose theelucidation of the epitope bound by a number of anti-VISTA antibodiesaccording to the invention. Thus, in exemplary embodiments agonistanti-human VISTA antibodies according to the invention will compriseIgG2 constant regions or fragments thereof, of the A form, B form or amixture of the foregoing. In exemplary embodiments these antibodies willbind to one or more FcγRs, e.g., they will bind to the same FcγRs as anintact or wild-type human IgG2 Fc region. In other exemplary embodimentsthe antibody will bind to CD32 (CD32A and/or CD32B). This may beaccomplished by the use of wild-type or modified IgG2 constant regionswhich bind to CD32 (CD32A and/or CD32B). Further, the agonist antibodymay be modified to incorporate another polypeptide such as another Fcpolypeptide or antigen binding region which binds to FcγRs such as CD32Aand/or CD32B.

The IgG2 Fc or constant regions contained in the inventive agonistanti-human VISTA antibodies optionally may be modified, e.g., in orderto alter effector function, e.g., to alter FcR binding, FcN binding,complement binding, glycosylation and the like. In particular, the IgG2Fc or constant regions contained in the inventive agonist anti-humanVISTA antibodies optionally may be modified by the conversion of thecysteine at position 27 or further optionally by the conversion ofanother cysteine residue or other residues, e.g., in the hinge region toanother amino acid, e.g., a serine. Other potential Fc modifications aredisclosed infra.

These VISTA agonist antibodies may be used in treating or preventingdiseases conditions or for treating or reducing, ameliorating thepathological effects associated therewith, e.g., inflammation, intreating or preventing conditions wherein the suppression of T cellimmunity or the expression of proinflammatory cytokines and or increasedexpression of chemokines and chemoattractants is therapeutically orprophylactically beneficial. These conditions include in particularautoimmunity, allergy, inflammatory disorders, sepsis, GVHD and forinhibiting unwanted T cell immune responses against transplanted cells,tissues or organs such as CAR-T cell or gene therapy constructs or cellscontaining.

As mentioned exemplary conditions which may be treated therapeuticallyor prophylactically using an agonist anti-human VISTA antibody accordingto the invention include autoimmune conditions, allergy conditions,inflammatory conditions, GVHD, transplant and sepsis. As mentioned,agonist anti-human VISTA antibodies according to the invention have beenshown to be therapeutically effective and to be immunosuppressive innumerous animal disease models including arthritis, inflammatory boweldisease (IBD), lupus, GVHD, chronic acute infection/hepatotoxicity andpsoriasis disease models. Therefore the inventive antibodies should bewell suited for use in treating conditions wherein the suppression ofimmunity, especially T cell immunity is therapeutically desired.

A. Use of Agonistic Anti-Human Vista Antibodies and Fragments in Therapyand Diagnosis

Compositions containing agonists according to the invention may be usedto inhibit T cell immunity and to treat conditions where this istherapeutically desirable such as autoimmunity, allergy or inflammatoryconditions. These compositions will comprise an amount of an agonistantibody or antibody fragment according to the invention effective tosuppress T cell activation or proliferation or cytokine expression orother effects of VISTA in a subject in need thereof. Such autoimmune,inflammatory and allergic conditions include for example arthriticconditions such as RA, psoriatic arthritis, psoriasis, scleroderma,multiple sclerosis, lupus, IBD, ITP, diabetes, GVHD, sarcoidosis,allergic asthma, hepatitis associated hepatotoxicity and for inhibitingunwanted T cell immune responses against transplanted cells, tissues ororgans such as CAR-T cell or gene therapy constructs or cells containingand the like.

Specific conditions wherein the inventive antibodies may be used aloneor in association with other therapeutics, especially otherimmunosuppressant molecules include acquired immune deficiency syndrome(AIDS), acquired splenic atrophy, acute anterior uveitis, AcuteDisseminated Encephalomyelitis (ADEM), acute gouty arthritis, acutenecrotizing hemorrhagic leukoencephalitis, acute or chronic sinusitis,acute purulent meningitis (or other central nervous system inflammatorydisorders), acute serious inflammation, Addison's disease, adrenalitis,adult onset diabetes mellitus (Type II diabetes), adult-onset idiopathichypoparathyroidism (AOIH), Agammaglobulinemia, agranulocytosis,vasculitides, including vasculitis, optionally, large vessel vasculitis,optionally, polymyalgia rheumatica and giant cell (Takayasu's)arthritis, allergic conditions, allergic contact dermatitis, allergicdermatitis, allergic granulomatous angiitis, allergic hypersensitivitydisorders, allergic neuritis, allergic reaction, alopecia areata,alopecia totalis, Alport's syndrome, alveolitis, optionally allergicalveolitis or fibrosing alveolitis, Alzheimer's disease, amyloidosis,amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), aneosinophil-related disorder, optionally eosinophilia, anaphylaxis,ankylosing spondylitis, angiectasis, antibody-mediated nephritis,Anti-GBM/Anti-TBM nephritis, antigen-antibody complex-mediated diseases,antiglomerular basement membrane disease, antiphospholipid antibodysyndrome, antiphospholipid syndrome (APS), aphthae, aphthous stomatitis,aplastic anemia, arrhythmia, arteriosclerosis, arterioscleroticdisorders, arthritis, optionally rheumatoid arthritis such as acutearthritis, or chronic rheumatoid arthritis, arthritis chronicaprogrediente, arthritis deformans, ascariasis, aspergilloma, granulomascontaining eosinophils, aspergillosis, aspermiogenese, asthma,optionally asthma bronchiale, bronchial asthma, or auto-immune asthma,ataxia telangiectasia, ataxic sclerosis, atherosclerosis, autism,autoimmune angioedema, autoimmune aplastic anemia, autoimmune atrophicgastritis, autoimmune diabetes, autoimmune disease of the testis andovary including autoimmune orchitis and oophoritis, autoimmune disordersassociated with collagen disease, autoimmune dysautonomia, autoimmuneear disease, optionally autoimmune inner ear disease (AGED), autoimmuneendocrine diseases including thyroiditis such as autoimmune thyroiditis,autoimmune enteropathy syndrome, autoimmune gonadal failure, autoimmunehearing loss, autoimmune hemolysis, Autoimmune hepatitis, autoimmunehepatological disorder, autoimmune hyperlipidemia, autoimmuneimmunodeficiency, autoimmune inner ear disease (AIED), autoimmunemyocarditis, autoimmune neutropenia, autoimmune pancreatitis, autoimmunepolyendocrinopathies, autoimmune polyglandular syndrome type I,autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP),autoimmune thyroid disease, autoimmune urticaria, autoimmune-mediatedgastrointestinal diseases, Axonal & neuronal neuropathies, Balo disease,Behçet's disease, benign familial and ischemia-reperfusion injury,benign lymphocytic angiitis, Berger's disease (IgA nephropathy),bird-fancier's lung, blindness, Boeck's disease, bronchiolitisobliterans (non-transplant) vs NSIP, bronchitis, bronchopneumonicaspergillosis, Bruton's syndrome, bullous pemphigoid, Caplan's syndrome,Cardiomyopathy, cardiovascular ischemia, Castleman's syndrome, Celiacdisease, celiac sprue (gluten enteropathy), cerebellar degeneration,cerebral ischemia, and disease accompanying vascularization, Chagasdisease, channelopathies, optionally epilepsy, channelopathies of theCNS, chorioretinitis, choroiditis, an autoimmune hematological disorder,chronic active hepatitis or autoimmune chronic active hepatitis, chroniccontact dermatitis, chronic eosinophilic pneumonia, chronic fatiguesyndrome, chronic hepatitis, chronic hypersensitivity pneumonitis,chronic inflammatory arthritis, Chronic inflammatory demyelinatingpolyneuropathy (CIDP), chronic intractable inflammation, chronicmucocutaneous candidiasis, chronic neuropathy, optionally IgMpolyneuropathies or IgM-mediated neuropathy, chronic obstructive airwaydisease, chronic pulmonary inflammatory disease, Chronic recurrentmultifocal osteomyelitis (CRMO), chronic thyroiditis (Hashimoto'sthyroiditis) or subacute thyroiditis, Churg-Strauss syndrome,cicatricial pemphigoid/benign mucosal pemphigoid, CNS inflammatorydisorders, CNS vasculitis, Coeliac disease, Cogan's syndrome, coldagglutinin disease, colitis polyposa, colitis such as ulcerativecolitis, colitis ulcerosa, collagenous colitis, conditions involvinginfiltration of T cells and chronic inflammatory responses, congenitalheart block, congenital rubella infection, Coombs positive anemia,coronary artery disease, Coxsackie myocarditis, CREST syndrome(calcinosis, Raynaud's phenomenon), Crohn's disease, cryoglobulinemia,Cushing's syndrome, cyclitis, optionally chronic cyclitis, heterochroniccyclitis, iridocyclitis, or Fuch's cyclitis, cystic fibrosis,cytokine-induced toxicity, deafness, degenerative arthritis,demyelinating diseases, optionally autoimmune demyelinating diseases,demyelinating neuropathies, dengue, dermatitis herpetiformis and atopicdermatitis, dermatitis including contact dermatitis, dermatomyositis,dermatoses with acute inflammatory components, Devic's disease(neuromyelitis optica), diabetic large-artery disorder, diabeticnephropathy, diabetic retinopathy, Diamond Blackfan anemia, diffuseinterstitial pulmonary fibrosis, dilated cardiomyopathy, discoid lupus,diseases involving leukocyte diapedesis, Dressler's syndrome,Dupuytren's contracture, echovirus infection, eczema including allergicor atopic eczema, encephalitis such as Rasmussen's encephalitis andlimbic and/or brainstem encephalitis, encephalomyelitis, optionallyallergic encephalomyelitis or encephalomyelitis allergica andexperimental allergic encephalomyelitis (EAE), endarterial hyperplasia,endocarditis, endocrine ophthalmopathy, endometriosis, endomyocardialfibrosis, endophthalmia phacoanaphylactica, endophthalmitis, enteritisallergica, eosinophilia-myalgia syndrome, eosinophilic fascitis,epidemic keratoconjunctivitis, epidermolysis bullosa acquisita (EBA),episclera, episcleritis, Epstein-Barr virus infection, erythema elevatumet diutinum, erythema multiforme, erythema nodosum leprosum, erythemanodosum, erythroblastosis fetalis, esophageal dysmotility, Essentialmixed cryoglobulinemia, ethmoid, Evan's syndrome, Experimental AllergicEncephalomyelitis (EAE), Factor VIII deficiency, farmer's lung, febrisrheumatica, Felty's syndrome, fibromyalgia, fibrosing alveolitis,filariasis, focal segmental glomerulosclerosis (FSGS), food poisoning,frontal, gastric atrophy, giant cell arthritis (temporal arthritis),giant cell hepatitis, giant cell polymyalgia, glomerulonephritides,glomerulonephritis (GN) with and without nephrotic syndrome such aschronic or acute glomerulonephritis (e.g., primary GN), Goodpasture'ssyndrome, gouty arthritis, granulocyte transfusion-associated syndromes,granulomatosis including lymphomatoid granulomatosis, granulomatosiswith polyangiitis (GPA), granulomatous uveitis, Grave's disease,Guillain-Barre syndrome, gutatte psoriasis, hemoglobinuriaparoxysmatica, Hamman-Rich's disease, Hashimoto's disease, Hashimoto'sencephalitis, Hashimoto's thyroiditis, hemochromatosis, hemolytic anemiaor immune hemolytic anemia including autoimmune hemolytic anemia (AIHA),hemolytic anemia, hemophilia A, Henoch-Schönlein purpura, Herpesgestationis, human immunodeficiency virus (HIV) infection, hyperalgesia,hypogammaglobulinemia, hypogonadism, hypoparathyroidism, idiopathicdiabetes insipidus, idiopathic facial paralysis, idiopathichypothyroidism, idiopathic IgA nephropathy, idiopathic membranous GN oridiopathic membranous nephropathy, idiopathic nephritic syndrome,idiopathic pulmonary fibrosis, idiopathic sprue, Idiopathicthrombocytopenic purpura (ITP), IgA nephropathy, IgE-mediated diseases,optionally anaphylaxis and allergic or atopic rhinitis, IgG4-relatedsclerosing disease, ileitis regionalis, immune complex nephritis, immuneresponses associated with acute and delayed hypersensitivity mediated bycytokines and T-lymphocytes, immune-mediated GN, immunoregulatorylipoproteins, including adult or acute respiratory distress syndrome(ARDS), Inclusion body myositis, infectious arthritis, infertility dueto antispermatozoan antibodies, inflammation of all or part of the uvea,inflammatory bowel disease (IBD) inflammatory hyperproliferative skindiseases, inflammatory myopathy, insulin-dependent diabetes (type 1),insulitis, Interstitial cystitis, interstitial lung disease,interstitial lung fibrosis, iritis, ischemic reperfusion disorder, jointinflammation, Juvenile arthritis, juvenile dermatomyositis, juvenilediabetes, juvenile onset (Type I) diabetes mellitus, including pediatricinsulin-dependent diabetes mellitus (IDDM), juvenile-onset rheumatoidarthritis, Kawasaki syndrome, keratoconjunctivitis sicca,kypanosomiasis, Lambert-Eaton syndrome, leishmaniasis, leprosy,leucopenia, leukocyte adhesion deficiency, Leukocytoclastic vasculitis,leukopenia, lichen planus, lichen sclerosus, ligneous conjunctivitis,linear IgA dermatosis, Linear IgA disease (LAD), Loffler's syndrome,lupoid hepatitis, lupus (including nephritis, cerebritis, pediatric,non-renal, extra-renal, discoid, alopecia), Lupus (SLE), lupuserythematosus disseminatus, Lyme arthritis, Lyme disease, lymphoidinterstitial pneumonitis, malaria, male and female autoimmuneinfertility, maxillary, medium vessel vasculitis (including Kawasaki'sdisease and polyarteritis nodosa), membrano- or membranous proliferativeGN (MPGN), including Type I and Type II, and rapidly progressive GN,membranous GN (membranous nephropathy), Meniere's disease, meningitis,microscopic colitis, microscopic polyangiitis, migraine, minimal changenephropathy, Mixed connective tissue disease (MCTD), mononucleosisinfectiosa, Mooren's ulcer, Mucha-Habermann disease, multifocal motorneuropathy, multiple endocrine failure, multiple organ injury syndromesuch as those secondary to septicemia, trauma or hemorrhage, multipleorgan injury syndrome, multiple sclerosis (MS) such as spino-optical MS,multiple sclerosis, mumps, muscular disorders, myasthenia gravis such asthymoma-associated myasthenia gravis, myasthenia gravis, myocarditis,myositis, narcolepsy, necrotizing enterocolitis, and transmural colitis,and autoimmune inflammatory bowel disease, necrotizing, cutaneous, orhypersensitivity vasculitis, neonatal lupus syndrome (NLE), nephrosis,nephrotic syndrome, neurological disease, neuromyelitis optica(Devic's), neuromyelitis optica, neuromyotonia, neutropenia,non-cancerous lymphocytosis, nongranulomatous uveitis, non-malignantthymoma, ocular and orbital inflammatory disorders, ocular cicatricialpemphigoid, oophoritis, ophthalmia symphatica, opsoclonus myoclonussyndrome (OMS), opsoclonus or opsoclonus myoclonus syndrome (OMS), andsensory neuropathy, optic neuritis, orchitis granulomatosa,osteoarthritis, palindromic rheumatism, pancreatitis, pancytopenia,PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated withStreptococcus), paraneoplastic cerebellar degeneration, paraneoplasticsyndrome, paraneoplastic syndromes, including neurologic paraneoplasticsyndromes, optionally Lambert-Eaton myasthenic syndrome or Eaton-Lambertsyndrome, parasitic diseases such as Leishmania, paroxysmal nocturnalhemoglobinuria (PNH), Parry Romberg syndrome, pars planitis (peripheraluveitis), Parsonnage-Turner syndrome, parvovirus infection, pemphigoidsuch as pemphigoid bullous and skin pemphigoid, pemphigus (includingpemphigus vulgaris), pemphigus erythematosus, pemphigus foliaceus,pemphigus mucus-membrane pemphigoid, pemphigus, peptic ulcer, periodicparalysis, peripheral neuropathy, perivenous encephalomyelitis,pernicious anemia (anemia perniciosa), pernicious anemia, phacoantigenicuveitis, pneumonocirrhosis, POEMS syndrome, polyarteritis nodosa, TypeI, II, & III, polyarthritis chronica primaria, polychondritis (e.g.,refractory or relapsed polychondritis), polyendocrine autoimmunedisease, polyendocrine failure, polyglandular syndromes, optionallyautoimmune polyglandular syndromes (or polyglandular endocrinopathysyndromes), polymyalgia rheumatica, polymyositis,polymyositis/dermatomyositis, polyneuropathies, polyradiculitis acuta,post-cardiotomy syndrome, posterior uveitis, or autoimmune uveitis,postmyocardial infarction syndrome, postpericardiotomy syndrome,post-streptococcal nephritis, post-vaccination syndromes, preseniledementia, primary biliary cirrhosis, primary hypothyroidism, primaryidiopathic myxedema, primary lymphocytosis, which includes monoclonal Bcell lymphocytosis, optionally benign monoclonal gammopathy andmonoclonal garnmopathy of undetermined significance, MGUS, primarymyxedema, primary progressive MS (PPMS), and relapsing remitting MS(RRMS), primary sclerosing cholangitis, progesterone dermatitis,progressive systemic sclerosis, proliferative arthritis, psoriasis suchas plaque psoriasis, psoriasis, psoriatic arthritis, pulmonary alveolarproteinosis, pulmonary infiltration eosinophilia, pure red cell anemiaor aplasia (PRCA), pure red cell aplasia, purulent or nonpurulentsinusitis, pustular psoriasis and psoriasis of the nails, pyelitis,pyoderma gangrenosum, Quervain's thyroiditis, Raynaud's phenomenon,reactive arthritis, recurrent abortion, reduction in blood pressureresponse, reflex sympathetic dystrophy, refractory sprue, Reiter'sdisease or syndrome, relapsing polychondritis, reperfusion injury ofmyocardial or other tissues, reperfusion injury, respiratory distresssyndrome, restless legs syndrome, retinal autoimmunity, retroperitonealfibrosis, Reynaud's syndrome, rheumatic diseases, rheumatic fever,rheumatism, rheumatoid arthritis, rheumatoid spondylitis, rubella virusinfection, Sampter's syndrome, sarcoidosis, schistosomiasis, Schmidtsyndrome, SCID and Epstein-Barr virus-associated diseases, sclera,scleritis, sclerodactyl, scleroderma, optionally systemic scleroderma,sclerosing cholangitis, sclerosis disseminata, sclerosis such assystemic sclerosis, sensoneural hearing loss, seronegativespondyloarthritides, Sheehan's syndrome, Shulman's syndrome, silicosis,Sjögren's syndrome, sperm & testicular autoimmunity, sphenoid sinusitis,Stevens-Johnson syndrome, stiff-man (or stiff-person) syndrome, subacutebacterial endocarditis (SBE), subacute cutaneous lupus erythematosus,sudden hearing loss, Susac's syndrome, Sydenham's chorea, sympatheticophthalmia, systemic lupus erythematosus (SLE) or systemic lupuserythematodes, cutaneous SLE, systemic necrotizing vasculitis,ANCA-associated vasculitis, optionally Churg-Strauss vasculitis orsyndrome (CSS), tabes dorsalis, Takayasu's arteritis, telangiectasia,temporal arteritis/Giant cell arteritis, thromboangiitis ubiterans,thrombocytopenia, including thrombotic thrombocytopenic purpura (TTP)and autoimmune or immune-mediated thrombocytopenia such as idiopathicthrombocytopenic purpura (ITP) including chronic or acute ITP,thrombocytopenic purpura (TTP), thyrotoxicosis, tissue injury,Tolosa-Hunt syndrome, toxic epidermal necrolysis, toxic-shock syndrome,transfusion reaction, transient hypogammaglobulinemia of infancy,transverse myelitis, traverse myelitis, tropical pulmonary eosinophilia,tuberculosis, ulcerative colitis, undifferentiated connective tissuedisease (UCTD), urticaria, optionally chronic allergic urticaria andchronic idiopathic urticaria, including chronic autoimmune urticaria,uveitis, anterior uveitis, uveoretinitis, valvulitis, vasculardysfunction, vasculitis, vertebral arthritis, vesiculobullousdermatosis, vitiligo, Wegener's granulomatosis (Granulomatosis withPolyangiitis (GPA)), Wiskott-Aldrich syndrome, or x-linked hyper IgMsyndrome.

It should be understood that the disease conditions identified hereinare intended to be exemplary and not exhaustive.

The subject agonists may be combined with other therapeutics which maybe administered in the same or different compositions, at the same ordifferent time and in either order. For example, the subject agonistsmay be administered in a therapeutic regimen that includes theadministration of a PD-1 or PD-L1 agonist, CTLA4-Ig, a cytokine, acytokine agonist or antagonist, or another receptor agonist orantagonist.

Downregulation of Immune Responses

Upregulating or enhancing the inhibitory function of a VISTA polypeptidemay be used to downregulate immune responses. Downregulation can be inthe form of inhibiting or blocking an immune response already inprogress, or may involve preventing the induction of an immune response.The functions of activated immune cells can be inhibited bydownregulating immune cell responses or by inducing specific anergy inimmune cells, or both. For example, VISTA agonist antibodies may bind tothe VISTA polypeptide which is expressed on various immune cells andthereby downmodulate the immune response. This agonist antibody may bemonospecific or multispecific, e.g., it may comprise a bispecificantibody such as a BiTE. For example, such an antibody can comprise aVISTA antigen binding moiety and another antigen binding moiety, e.g.,which targets a cell surface receptor on an immune cell, e.g., a T cell,a B cell, or a myeloid cell. Such an antibody, in addition to comprisinga VISTA antigen binding site, may comprise a binding site which binds toa B cell antigen receptor, a T cell antigen receptor, or an Fc or otherreceptor, in order to target the molecule to a specific cell population.Selection of this second antigen for the bispecific antibody providesflexibility in selection of cell population to be targeted. VISTAagonist antibodies that promote or mimic VISTA activity may enhance theinteraction of VISTA with its natural binding partners. As disclosedherein other human VISTA activating or agonist antibodies can beidentified by their ability to inhibit T cell activity or proliferationand/or based on their immunosuppressive effects in vitro orinflammatory, allergic or autoimmune disease models.

A number of art-recognized readouts of cell activation can be employedto measure, e.g., cell proliferation or effector function (e.g.,antibody production, cytokine production, phagocytosis) in the presenceof the activating agent. The ability of a test antibody to agonize orpromote the effects of human VISTA and thereby block this activation canbe readily determined by measuring the ability of the agent to affect adecrease in proliferation or effector function being measured.Accordingly, the ability of a test antibody to be immunosuppressive andto block immune activation can be determined by measuring cytokineproduction and/or proliferation at different concentrations of antigen.

Tolerance may be induced against specific antigens by co-administeringan antigen with a VISTA agonist antibody according to the invention. Forexample, tolerance may be induced to specific polypeptides Immuneresponses to allergens or foreign polypeptides to which an immuneresponse is undesirable can be inhibited. For example, patients thatreceive Factor VIII frequently generate antibodies against this clottingfactor. Co-administration of a VISTA agonist antibody according to theinvention that stimulates or mimics VISTA activity or interaction withits natural binding partner, with recombinant factor VIII may suppressthis undesired immune response.

A VISTA agonist antibody according to the invention may be used incombination with another agent that blocks the activity of costimulatoryreceptors on an immune cell or which agonizes the activity of anotherimmunosuppressive receptor or ligand expressed on immune cells in orderto downmodulate immune responses. Exemplary molecules include: PD-1,PDL-1 agonists, soluble forms of CTLA-4, anti-B7-1 antibodies, anti-B7-2antibodies, antagonistic antibodies targeting one or more of LAG-3,TIM-3, BTLA, B7-H4, B7H3, et al. and/or agonistic antibodies targetingone or more of CD40, CD137, OX40, GITR, CD27, CD28 or ICOS orcombinations thereof. These moieties can be combined in a singlecomposition or compound, e.g., a bispecific antibody containing a VISTAagonist antibody according to the invention and further comprisinganother immune agonist antibody or it may comprise a fusion polypeptidecontaining a VISTA agonist antibody according to the invention which isfused to another immunosuppressive polypeptide or other active agent.Alternatively these moieties may be administered as separate or discreteentities (simultaneously or sequentially) in the same or differentcompositions to down regulate immune cell mediated immune responses in asubject.

Examples of specific immmunoinhibitory molecules that may be combinedwith VISTA agonist antibodies according to the invention includeantibodies that block a costimulatory signal (e.g., against CD28 orICOS), antibodies that activate an inhibitory signal via CTLA4, and/orantibodies against other immune cell markers (e.g., against CD40, CD40ligand, or cytokines), fusion proteins (e.g., CTLA4-Fc or PD-1-Fc), andimmunosuppressive drugs (e.g., rapamycin, cyclosporine A, or FK506).

In a further embodiment, bispecific antibodies containing VISTA agonistantibodies according to the invention are useful for targeting aspecific cell population, e.g., using a marker found only on a certaintype of cell, e.g., B lymphocytes, monocytes, dendritic cells, orLangerhans cells. Downregulating immune responses by activating VISTAactivity or VISTA-immune cell interactions (and thus stimulating thenegative signaling function of VISTA) is useful in downmodulating theimmune response, e.g., in situations of tissue, skin and organtransplantation, in graft-versus-host disease (GVHD), or allergies, orin autoimmune and inflammatory diseases such as systemic lupuserythematosus, IBD, RA, psoriasis and multiple sclerosis. For example,blockage of immune cell function results in reduced tissue destructionin tissue transplantation. Typically, in tissue transplants, rejectionof the transplant is initiated through its recognition as foreign byimmune cells, followed by an immune reaction that destroys thetransplant. The administration of a molecule which promotes the activityof VISTA or the interaction of VISTA with its natural bindingpartner(s), on immune cells alone or in conjunction with anotherdownmodulatory agent prior to or at the time of transplantation caninhibit the generation of a costimulatory signal. Moreover, promotion ofVISTA activity may also be sufficient to anergize the immune cells,thereby inducing tolerance in a subject.

To achieve sufficient immunosuppression or tolerance in some diseases orin some subjects, it may necessary to block the costimulatory functionof other molecules. For example, it may be desirable to block thefunction of B7-1 and B7-2 by administering a soluble form of acombination of peptides having an activity of each of these antigens orblocking antibodies against these antigens (separately or together in asingle composition) prior to or at the time of transplantation.Alternatively, it may be desirable to promote inhibitory activity ofVISTA and to further inhibit a costimulatory activity of B7-1 and/orB7-2.

The subject anti-human VISTA agonist antibodies are especially useful intreating autoimmune disease. Many autoimmune disorders are the result ofinappropriate activation of immune cells that are reactive againstself-tissue and which promote the production of cytokines andautoantibodies involved in the pathology of the diseases. Preventing theactivation of autoreactive immune cells may reduce or eliminate diseasesymptoms. Administration of the subject anti-human VISTA agonistantibodies that promote activity of VISTA or VISTA interaction with itsnatural binding partner(s), may induce antigen-specific tolerance ofautoreactive immune cells which could lead to long-term relief from thedisease. Additionally, co-administration of agents which blockcostimulation of immune cells by disrupting receptor-ligand interactionsof B7 molecules with costimulatory receptors may be useful in inhibitingimmune cell activation to prevent production of autoantibodies orcytokines which may be involved in the disease process.

Downregulation of an immune response via stimulation of VISTA activityor VISTA interaction with its natural binding partner(s) using thesubject anti-human VISTA agonist antibodies may also be useful intreating an autoimmune attack of autologous tissues. Thus, conditionsthat are caused or exacerbated by autoimmune attack (e.g., heartdisease, myocardial infarction or atherosclerosis) may be ameliorated orimproved by increasing VISTA activity or VISTA binding to its naturalbinding partner. It is therefore within the scope of the invention tomodulate conditions exacerbated by autoimmune attack, such as autoimmunedisorders (as well as conditions such as heart disease, myocardialinfarction, and atherosclerosis) by stimulating VISTA activity or VISTAinteraction with its counter receptor using the subject anti-human VISTAagonist antibodies.

As mentioned previously the efficacy of agonist anti-human VISTAantibodies according to the invention for preventing or alleviatingautoimmune and inflammatory disorders can be determined using a numberof well-characterized animal models of human autoimmune and inflammatorydiseases. Examples include murine experimental autoimmune encephalitis,systemic lupus erythematosus in MRL/Ipr/Ipr mice or NZB hybrid mice,murine autoimmune collagen arthritis, diabetes mellitus in NOD mice andBB rats, and murine experimental myasthenia gravis. See Paul ed.,Fundamental Immunology, Raven Press, New York, 1989, pages 840-856.

Inhibition of immune cell activation is further useful therapeuticallyin the treatment of allergies and allergic reactions, e.g., byinhibiting IgE production. The subject anti-human VISTA agonistantibodies which promote or mimic VISTA activity or VISTA interactionwith its natural binding partner(s) can be administered to an allergicsubject to inhibit immune cell-mediated allergic responses in thesubject. Stimulation of VISTA activity or interaction with its naturalbinding partner(s), can be accompanied by exposure to allergen inconjunction with appropriate MHC molecules. Allergic reactions can besystemic or local in nature, depending on the route of entry of theallergen and the pattern of deposition of IgE on mast cells orbasophils. Thus, immune cell-mediated allergic responses can beinhibited locally or systemically by administration of the subjectanti-human VISTA agonist antibodies.

Selection of Anti-VISTA Antibodies that Bind to the Same Epitope

In certain embodiments, an agonistic anti-VISTA antibody according tothe invention possesses desired functional properties such as modulationof immune stimulation and related functions. As shown in FIG. 4 anddisclosed in the working examples, the epitopic specificity of a numberof anti-human VISTA agonist antibodies according to the invention hasbeen elucidated. As a number of antibodies which have been shown to bindto the same epitope have been found to be immunosuppressive it isexpected that other VISTA agonist antibodies may be identified whichbind to the same or overlapping epitope, i.e., they will interact withone or more of the amino acid residues of human VISTA polypeptide withwhich the exemplary VISTA agonist antibodies bind. Other antibodies withthe same epitopic specificity may be selected and/or those which havethe ability to cross-compete for binding to VISTA antigen with thedesired antibodies. For example, the epitopic specificity of a desiredantibody may be determined using a library of overlapping peptidescomprising the entire VISTA polypeptide, e.g., 15-mers or an overlappingpeptide library constituting a portion containing a desired epitope ofVISTA and antibodies which bind to the same peptides or one or moreresidues thereof in the library are determined to bind the same linearor conformational epitope. In the examples the epitopic specificity wasdetermined using Pepscan® methods which may be used to identify linearand conformational epitopes.

Modification of Agonist Antibodies According to the Invention

In addition or alternative to modifications made within the framework orCDR regions, antibodies according to at least some embodiments of theinvention may be engineered to include modifications within the Fcregion, typically to alter one or more functional properties of theantibody, such as serum half-life, complement fixation, Fc receptorbinding, and/or antigen-dependent cellular cytotoxicity. Furthermore, anantibody according to at least some embodiments of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Suchembodiments are described further below. The numbering of residues inthe Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CHI is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcal protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S, andT256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half-life, the antibody can be altered withinthe CH1 or C_(L) region to contain a salvage receptor binding epitopetaken from two loops of a CH2 domain of an Fc region of an IgG, asdescribed in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidsselected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and322 can be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the CIcomponent of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551 by Idusogie etal.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351 by Bodmer et al.

In yet another example, the Fc region is modified to increase theaffinity of the antibody for an Fγ receptor by modifying one or moreamino acids at the following positions: 238, 239, 248, 249, 252, 254,255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285,286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309,312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337,338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430,434, 435, 437, 438 or 439. This approach is described further in PCTPublication WO 00/42072 by Presta. Moreover, the binding sites on humanIgGI for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variantswith improved binding have been described (see Shields, R. L. et al.(2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions256, 290, 298, 333, 334 and 339 are shown to improve binding to FcyRIII.Additionally, the following combination mutants are shown to improveFcyRIII binding: T256A/S298A, S298A/E333A, S298A/K224A andS298A/E333A/K334A. Furthermore, mutations such as M252Y/S254T/T256E orM428L/N434S improve binding to FcRn and increase antibody circulationhalf-life (see Chan C A and Carter P J (2010) Nature Rev Immunol10:301-316).

In still another embodiment, the antibody can be modified to abrogate invivo Fab arm exchange. Specifically, this process involves the exchangeof IgG4 half-molecules (one heavy chain plus one light chain) betweenother IgG4 antibodies that effectively results in b specific antibodieswhich are functionally monovalent. Mutations to the hinge region andconstant domains of the heavy chain can abrogate this exchange (seeAalberse, R C, Schuurman J., 2002, Immunology 105:9-19).

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycosylated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglyclosylationmay increase the affinity of the antibody for antigen. Such an approachis described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861by Co et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies according to at least some embodiments of theinvention to thereby produce an antibody with altered glycosylation. Forexample, the cell lines Ms704, Ms705, and Ms709 lack thefucosyltransferase gene, FUT8 (a (1,6) fucosyltransferase), such thatantibodies expressed in the Ms704, Ms705, and Ms709 cell lines lackfucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8 celllines are created by the targeted disruption of the FUT8 gene inCHO/DG44 cells using two replacement vectors (see U.S. PatentPublication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al.(2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 byHanai et al. describes a cell line with a functionally disrupted FUT8gene, which encodes a fucosyl transferase, such that antibodiesexpressed in such a cell line exhibit hypofucosylation by reducing oreliminating the a 1,6 bond-related enzyme. Hanai et al. also describecell lines which have a low enzyme activity for adding fucose to theN-acetylglucosamine that binds to the Fc region of the antibody or doesnot have the enzyme activity, for example the rat myeloma cell lineYB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 by Presta describesa variant CHO cell line, Lecl3 cells, with reduced ability to attachfucose to Asn(297)-linked carbohydrates, also resulting inhypofucosylation of antibodies expressed in that host cell (see alsoShields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740). PCTPublication WO 99/54342 by Umana et al. describes cell lines engineeredto express glycoprotein-modifying glycosyl transferases (e.g.,P(1,4)-N-acetylgl ucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17: 176-180).Alternatively, the fucose residues of the antibody may be cleaved offusing a fucosidase enzyme. For example, the fucosidase α-L-fucosidaseremoves fucosyl residues from antibodies (Tarentino, A. L. et al. (1975)Biochem. 14:5516-23).

Another modification of the antibodies herein that is contemplated bythe invention is pegylation or the addition of other water solublemoieties, typically polymers, e.g., in order to enhance half-life. Anantibody can be pegylated to, for example, increase the biological(e.g., serum) half-life of the antibody. To pegylate an antibody, theantibody, or fragment thereof, typically is reacted with polyethyleneglycol (PEG), such as a reactive ester or aldehyde derivative of PEG,under conditions in which one or more PEG groups become attached to theantibody or antibody fragment. Preferably, the pegylation is carried outvia an acylation reaction or an alkylation reaction with a reactive PEGmolecule (or an analogous reactive water-soluble polymer). As usedherein, the term “polyethylene glycol” is intended to encompass any ofthe forms of PEG that have been used to derivatize other proteins, suchas mono (Ci-Cio) alkoxy- or aryloxy-polyethylene glycol or polyethyleneglycol-maleimide. In certain embodiments, the antibody to be pegylatedis an aglycosylated antibody. Methods for pegylating proteins are knownin the art and can be applied to the antibodies according to at leastsome embodiments of the invention. See for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

Methods of Engineering Antibodies

In certain embodiments, an agonist anti-VISTA antibody according to theinvention having V_(H) and V_(L) sequences can be used to create newanti-VISTA antibodies, respectively, by modifying the V_(H) and/or V_(L)sequences, or the constant regions attached thereto. Thus, in anotheraspect according to at least some embodiments of the invention, thestructural features of an anti-VISTA antibody according to at least someembodiments of the invention, are used to create structurally relatedanti-VISTA antibodies that retain at least one functional property ofthe antibodies according to at least some embodiments of the invention,such as binding to human VISTA. For example, one or more CDR regions ofone VISTA antibody or mutations thereof can be combined recombinantlywith known framework regions and/or other CDRs to create additional,recombinantly-engineered, anti-VISTA antibodies according to at leastsome embodiments of the invention, as discussed above. Other types ofmodifications include those described in the previous section. Thestarting material for the engineering method is one or more of the V_(I)and/or V_(L) sequences provided herein, or one or more CDR regionsthereof. To create the engineered antibody, it is not necessary toactually prepare (i.e., express as a protein) an antibody having one ormore of the V_(H) and/or V_(L) sequences provided herein, or one or moreCDR regions thereof. Rather, the information contained in the sequencesis used as the starting material to create a “second generation”sequences derived from the original sequences and then the “secondgeneration” sequences is prepared and expressed as a protein.

Standard molecular biology techniques can be used to prepare and expressaltered antibody sequence. Preferably, the anti-VISTA antibody encodedby the altered antibody sequences is one that retains one, some or allof the functional properties of the anti-VISTA antibodies, respectively,produced by methods and with sequences provided herein, which functionalproperties include binding to VISTA antigen with a specific K_(D) levelor less and/or modulating immune responses and/or selectively binding todesired target cells such as for example, that express VISTA antigen.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein. Incertain embodiments of the methods of engineering antibodies accordingto at least some embodiments of the invention, mutations can beintroduced randomly or selectively along all or part of an anti-VISTAantibody coding sequence and the resulting modified anti-VISTAantibodies can be screened for binding activity and/or other desiredfunctional properties.

Mutational methods have been described in the art. For example, PCTPublication WO 02/092780 by Short describes methods for creating andscreening antibody mutations using saturation mutagenesis, syntheticligation assembly, or a combination thereof. Alternatively, PCTPublication WO 03/074679 by Lazar et al. describes methods of usingcomputational screening methods to optimize physiochemical properties ofantibodies.

Nucleic Acid Molecules Encoding Antibodies

The invention further provides nucleic acids which encode an anti-VISTAantibody according to the invention, or a fragment or conjugate thereof.The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art. See, F. Ausubel, etal., ed. (1987) Current Protocols in Molecular Biology, GreenePublishing and Wiley Interscience, New York. A nucleic acid according toat least some embodiments of the invention can be, for example, DNA orRNA and may or may not contain intronic sequences. In a preferredembodiment, the nucleic acid is a cDNA molecule.

Nucleic acids according to at least some embodiments of the inventioncan be obtained using standard molecular biology techniques. Forantibodies expressed by hybridomas (e.g., hybridomas prepared fromtransgenic mice carrying human immunoglobulin genes as described furtherbelow), cDNAs encoding the light and heavy chains of the antibody madeby the hybridoma can be obtained by standard PCR amplification or cDNAcloning techniques. For antibodies obtained from an immunoglobulin genelibrary (e.g., using phage display techniques), nucleic acid encodingthe antibody can be recovered from the library.

Once DNA fragments encoding V_(H) and V_(L) segments are obtained, theseDNA fragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a V_(L)- or V_(H)-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. As previouslydefined, “operatively linked” means that that the two DNA fragments arejoined such that the amino acid sequences encoded by the two DNAfragments remain in-frame.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH1,CH2 and CH3). The sequences of human heavy chain constant region genesare known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgGI, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgGI, IgG2 or IgG4 constant region. For a Fab fragment heavy chaingene, the VH-encoding DNA can be operatively linked to another DNAmolecule encoding only the heavy chain C_(H1) constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, C_(L)—The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabat,E. A., et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification. The light chain constantregion can be a kappa (κ) or lambda (λ) constant region, but mostpreferably is a K constant region.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly4-Ser)3, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (seee.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc.Natl. Acad. Sci., USA 85:5879-5883; McCafferty et al., (1990) Nature348:552-554).

Production of Anti-VISTA Monoclonal Antibodies

Anti-VISTA monoclonal antibodies (mAbs) and antigen-binding fragmentsaccording to the present invention can be produced by a variety oftechniques, including conventional monoclonal antibody methodology e.g.,the standard somatic cell hybridization technique of Kohler and Milstein(1975) Nature 256:495. Although somatic cell hybridization proceduresare preferred, in principle, other techniques for producing monoclonalantibody can be employed e.g., viral or oncogenic transformation of Blymphocytes.

A preferred animal system for preparing hybridomas is the murine system.Hybridoma production in the mouse is a very well-established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known. Chimeric orhumanized antibodies of the present invention can be prepared based onthe sequence of a murine monoclonal antibody prepared as describedabove. DNA encoding the heavy and light chain immunoglobulins can beobtained from the murine hybridoma of interest and engineered to containnon-murine (e.g., human) immunoglobulin sequences using standardmolecular biology techniques. For example, to create a chimericantibody, the murine variable regions can be linked to human constantregions using methods known in the art (see e.g., U.S. Pat. No.4,816,567 to Cabilly et al.). To create a humanized antibody, the murineCDR regions can be inserted into a human framework using methods knownin the art (see e.g., U.S. Pat. No. 5,225,539 to Winter and U.S. Pat.Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

According to at least some embodiments of the invention, the antibodiesare human monoclonal antibodies. Such human monoclonal antibodiesdirected against VISTA can be generated using transgenic ortranschromosomic mice carrying parts of the human immune system ratherthan the mouse system. These transgenic and transchromosomic miceinclude mice referred to herein as the HuMAb Mouse™ and KM Mouse™,respectively, and are collectively referred to herein as “human Igmice.” The HuMAb Mouse™ (Medarex Inc.) contains human immunoglobulingene miniloci that encode unrearranged human heavy μ and γ and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgG κmonoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N.(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). The preparationand use of the HuMab Mouse®, and the genomic modifications carried bysuch mice, is further described in Taylor, L. et al. (1992) NucleicAcids Research 20:6287-6295; Chen, J. et al. (1993) InternationalImmunology 5:647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA90:3720-3724; Choi et al. (1993) Nature Genetics 4: 117-123; Chen, J. etal. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol.152:2912-2920; Taylor, L. et al. (1994) International Immunology6:579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, the contents of all of which are hereby specificallyincorporated by reference in their entirety. See further, U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay;U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 toKorman et al.

In another embodiment, human antibodies according to at least someembodiments of the invention can be raised using a mouse that carrieshuman immunoglobulin sequences on transgenes and transchomosomes, suchas a mouse that carries a human heavy chain transgene and a human lightchain transchromosome. Such mice, referred to herein as “KM Mice™”, aredescribed in detail in PCT Publication WO 02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-VISTA antibodies according to at least some embodiments of theinvention. For example, an alternative transgenic system referred to asthe Xenomouse (Abgenix, Inc.) can be used; such mice are described in,for example, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6, 150,584and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-VISTA antibodies according to at least some embodiments of theinvention. For example, mice carrying both a human heavy chaintranschromosome and a human light chain transchromosome, referred to as“TC mice” can be used; such mice are described in Tomizuka et al. (2000)Proc. Natl. Acad Sci. USA 97:722-727. Furthermore, cows carrying humanheavy and light chain transchromosomes have been described in the art(Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be usedto raise anti-VISTA antibodies according to at least some embodiments ofthe invention.

Human monoclonal antibodies according to at least some embodiments ofthe invention can also be prepared using phage display methods forscreening libraries of human immunoglobulin genes. Such phage displaymethods for isolating human antibodies are established in the art. Seefor example: U.S. Pat. Nos. 5,223,409; 5,403,484; and U.S. Pat. No.5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 toDower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty etal.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313;6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies according to at least some embodiments ofthe invention can also be prepared using SCID mice into which humanimmune cells have been reconstituted such that a human antibody responsecan be generated upon immunization. Such mice are described in, forexample, U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.

Immunization of Human Ig Mice

In some embodiments human Ig mice are used to raise human anti-VISTAantibodies according to the invention, e.g., by immunizing such micewith a purified or enriched preparation of VISTA antigen and/orrecombinant VISTA, or VISTA fusion protein, as described by Lonberg, N.et al. (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996)Nature Biotechnology 14: 845-851; and PCT Publication WO 98/24884 and WO01/14424. Preferably, the mice will be 6-16 weeks of age upon the firstinfusion. For example, a purified or recombinant preparation (doseranging from 0.5-500 μg) of VISTA antigen can be used to immunize thehuman Ig mice intraperitoneally.

In general transgenic mice respond when initially immunizedintraperitoneally (IP) with antigen in complete Freund's adjuvant,followed by every other week IP immunizations (up to a total of 6) withantigen in incomplete Freund's adjuvant. However, adjuvants other thanFreund's are also found to be effective. In addition, whole cells in theabsence of adjuvant are found to be highly immunogenic. The immuneresponse can be monitored over the course of the immunization protocolwith plasma samples being obtained by retroorbital bleeds. The plasmacan be screened by ELISA (as described below), and mice with sufficienttiters of anti-VISTA human immunoglobulin can be used for fusions. Micecan be boosted intravenously with antigen 3 days before sacrifice andremoval of the spleen. It is expected that 2-3 fusions for eachimmunization may need to be performed. Between 6 and 24 mice aretypically immunized for each antigen. Usually both HCo7 and HCol2strains are used. In addition, both HCo7 and HCol2 transgene can be bredtogether into a single mouse having two different human heavy chaintransgenes (HCo7/HCol2). Alternatively or additionally, the KM Mouse™strain can be used. In an exemplary embodiment these mice will beengineered to selectively produce human IgG2 antibodies.

Generation of Hybridomas Producing Human Monoclonal Antibodies

In certain embodiments, hybridomas producing a human monoclonalanti-VISTA antibody according to the invention may be generated usingsplenocytes and/or lymph node cells from immunized mice can be isolatedand fused to an appropriate immortalized cell line, such as a mousemyeloma cell line. The resulting hybridomas can be screened for theproduction of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toone-sixth the numbers of P3X63-Ag8.653 nonsecreting mouse myeloma cells(ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2×10⁵in flat bottom microtiter plate, followed by a two week incubation inselective medium containing 20% fetal Clone Serum, 18% “653” conditionedmedia, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mMHEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/mlstreptomycin, 50 mg/ml gentamycin and IX HAT (Sigma; the HAT is added 24hours after the fusion). After approximately two weeks, cells can becultured in medium in which the HAT is replaced with HT. Individualwells can then be screened by ELISA for human monoclonal IgM and IgGantibodies. Once extensive hybridoma growth occurs, medium can beobserved usually after 10-14 days. The antibody secreting hybridomas canbe replated, screened again, and if still positive for human IgG, themonoclonal antibodies can be subcloned at least twice by limitingdilution. The stable subclones can then be cultured in vitro to generatesmall amounts of antibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-Sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

Generation of Transfectomas Producing Monoclonal Antibodies

In certain embodiments, an anti-VISTA antibody according to theinvention can be produced in a host cell transfectoma using, forexample, a combination of recombinant DNA techniques and genetransfection methods as is well known in the art (e.g., Morrison, S.(1985) Science 229: 1202). For example, to express the antibodies, orantibody fragments thereof, DNAs encoding partial or full-length lightand heavy chains, can be obtained by standard molecular biologytechniques (e.g., PCR amplification or cDNA cloning using a hybridomathat expresses the antibody of interest) and the DNAs can be insertedinto expression vectors such that the genes are operatively linked totranscriptional and translational control sequences. In this context,the term “operatively linked” is intended to mean that an antibody geneis ligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the C_(H) segments within the vectorand the V_(L) segment is operatively linked to the C_(L) segment withinthe vector. Additionally or alternatively, the recombinant expressionvector can encode a signal peptide that facilitates secretion of theantibody chain from a host cell. The antibody chain gene can be clonedinto the vector such that the signal peptide is linked in-frame to theamino terminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

Characterization of Antibody Binding to Antigen

In certain embodiments, the binding specificity of an agonisticanti-VISTA antibody according to the invention is determined by knownantibody binding assay techniques such as ELISA. In an exemplary ELISA,microtiter plates are coated with a purified antigen, herein VISTA at0.25 μg/ml in PBS, and then blocked with 5% bovine serum albumin in PBS.Dilutions of antibody (e.g., dilutions of plasma from-immunized mice)are added to each well and incubated for 1-2 hours at 37° C. The platesare washed with PBS/Tween and then incubated with secondary reagent(e.g., for human antibodies, a goat-anti-human IgG Fc-specificpolyclonal reagent) conjugated to alkaline phosphatase for 1 hour at 37°C. After washing, the plates are developed with pNPP substrate (1mg/ml), and analyzed at OD of 405-650. Preferably, mice which developthe highest titers will be used for fusions.

An ELISA assay as described above can also be used to screen forhybridomas that show positive reactivity with VISTA immunogen.Hybridomas that bind with high avidity to VISTA are subcloned andfurther characterized. One clone from each hybridoma, which retains thereactivity of the parent cells (by ELISA), can be chosen for making a5-10 vial cell bank stored at −140° C., and for antibody purification.

To purify anti-VISTA antibodies, selected hybridomas can be grown intwo-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-Sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected anti-VISTA monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, II.). Competition studies usingunlabeled monoclonal antibodies and biotinylated monoclonal antibodiescan be performed using VISTA coated-ELISA plates as described above.Biotinylated mAb binding can be detected with a strep-avidin-alkalinephosphatase probe.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed using reagents specific for antibodies of a particularisotype, e.g., IgG2's. For example, to determine the isotype of a humanmonoclonal antibody, wells of microtiter plates can be coated with ̂g/mlof anti-human immunoglobulin overnight at 4° C. After blocking with 1%BSA, the plates are reacted with 1 mug/ml or less of test monoclonalantibodies or purified isotype controls, at ambient temperature for oneto two hours. The wells can then be reacted with either human IgG1 orhuman IgM-specific alkaline phosphatase-conjugated probes. Plates aredeveloped and analyzed as described above.

Anti-VISTA human IgGs can be further tested for reactivity with VISTAantigen, respectively, by Western blotting. Briefly, VISTA antigen canbe prepared and subjected to sodium dodecyl sulfate polyacrylamide gelelectrophoresis. After electrophoresis, the separated antigens aretransferred to nitrocellulose membranes, blocked with 10% fetal calfserum, and probed with the monoclonal antibodies to be tested. Human IgGbinding can be detected using anti-human IgG alkaline phosphatase anddeveloped with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis,Mo.).

In another aspect, the present invention features antibody-drugconjugates (ADCs), consisting of an antibody (or antibody fragment suchas a single-chain variable fragment (scFv) linked to a payload drug(often cytotoxic). The antibody causes the ADC to bind to the targetcancer cells. Often the ADC is then internalized by the cell and thedrug is released into the cell. Because of the targeting, the sideeffects are lower and give a wider therapeutic window. Hydrophiliclinkers (e.g., PEG4Mal) help prevent the drug being pumped out ofresistant cancer cells through MDR (multiple drug resistance)transporters.

In another aspect, the present invention features immunoconjugatescomprising an anti-VISTA antibody, or a fragment thereof, conjugated toa therapeutic agent, such as a cytotoxin, a drug (e.g., animmunosuppressant) or a radiotoxin. Such conjugates are referred toherein as “immunoconjugates”. Immunoconjugates that include one or morecytotoxins are referred to as “immunotoxins.” A cytotoxin or cytotoxicagent includes any agent that is detrimental to (e.g., kills) cells.Examples include Taxol, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine,colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents alsoinclude, for example, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thiotepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Other examples of therapeutic cytotoxins that can be conjugated to anantibody according to at least some embodiments of the invention includeduocarmycins, calicheamicin, maytansines and auristatins, andderivatives thereof. An example of a calicheamicin antibody conjugate iscommercially available (Mylotarg™ Wyeth).

Cytotoxins can be conjugated to antibodies according to at least someembodiments of the invention using linker technology available in theart. Examples of linker types that have been used to conjugate acytotoxin to an antibody include, but are not limited to, hydrazones,thioethers, esters, disulfides and peptide-containing linkers. A linkercan be chosen that is, for example, susceptible to cleavage by low pHwithin the lysosomal compartment or susceptible to cleavage byproteases, such as proteases preferentially expressed in tumor tissuesuch as cathepsins (e.g., cathepsins B, C, D). For further discussion oftypes of cytotoxins, linkers and methods for conjugating therapeuticagents to antibodies, see also Saito, G. et al. (2003) Adv. Drug Deliv.Rev. 55: 199-215; Trail, P. A. et al. (2003) Cancer Immunol. Immunother.52:328-337; Payne, G. (2003) Cancer Cell 3:207-212; Allen, T. M. (2002)Nat. Rev. Cancer 2:750-763; Pastan, I. and Kreitman, R. J. (2002) Curr.Opin. Investig. Drugs 3: 1089-1091; Senter, P. D. and Springer, C. J.(2001) Adv. Drug Deliv. Rev. 53:247-264.

Antibodies of the present invention also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine 131, indium 111,yttrium 90 and lutetium 177. Methods for preparing radioimmunoconjugatesare established in the art.

Radioimmunoconjugates are commercially available, including Zevalin®(BiogenlDEC) and Bexxars®. (Corixa Pharmaceuticals), and similar methodscan be used to prepare radioimmunoconjugates using the antibodiesaccording to at least some embodiments of the invention.

The agonist anti-human VISTA antibodies and conjugates containingaccording to at least some embodiments of the invention can be used tomodify a given biological response, and the drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, anenzymatically active toxin, or active fragment thereof, such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor or interferon-γ; or, biological response modifierssuch as, for example, lymphokines, interleukin-1 (“IL-”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Carriers Of Cytotoxic Agents InCancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological AndClinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);“Analysis, Results, And Future Prospective Of The Therapeutic Use OfRadiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies ForCancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16(Academic Press 1985), and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62: 119-58(1982).

Bispecific Molecules

According to at least some embodiments the invention also encompassesmultispecific anti-VISTA agonist antibodies. Multispecific antibodiesare monoclonal antibodies that have binding specificities for at leasttwo different sites. In another aspect, the present invention featuresbispecific molecules comprising an anti-VISTA antibody, or a fragmentthereof, according to at least some embodiments of the invention. Anantibody according to at least some embodiments of the invention, orantigen-binding portions thereof, can be derivatized or linked toanother functional molecule, e.g., another peptide or protein (e.g.,another antibody or ligand for a receptor) to generate a bispecificmolecule that binds to at least two different binding sites or targetmolecules. The antibody according to at least some embodiments of theinvention may in fact be derivatized or linked to more than one otherfunctional molecule to generate multispecific molecules that bind tomore than two different binding sites and/or target molecules; suchmultispecific molecules are also intended to be encompassed by the term“bispecific molecule” as used herein. To create a bispecific moleculeaccording to at least some embodiments of the invention, an antibody canbe functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other bindingmolecules, such as another antibody, antibody fragment, peptide orbinding mimetic, such that a bispecific molecule results. In certainembodiments, one of the binding specificities of the bispecificantibodies is for VISTA and the other is for any other antigen. Incertain embodiments, bispecific antibodies may bind to two differentepitopes of VISTA. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express VISTA. Bispecific antibodies canbe prepared as full length antibodies or antibody fragments.

A bispecific antibody according to at least some embodiments of theinvention is an antibody which can bind simultaneously to two targetswhich are of different structure. Bispecific antibodies (bsAb) andbispecific antibody fragments (bsFab) according to at least someembodiments of the invention have at least one arm that specificallybinds to a B-cell antigen or epitope and at least one other arm thatspecifically binds a targetable conjugate.

According to at least some embodiments the invention encompasses also afusion antibody protein, which is a recombinantly producedantigen-binding molecule in which two or more different single-chainantibody or antibody fragment segments with the same or differentspecificities are linked. A variety of bispecific fusion antibodyproteins can be produced using molecular engineering. In one form, thebispecific fusion antibody protein is monovalent, consisting of, forexample, a sent with a single binding site for one antigen and a Fabfragment with a single binding site for a second antigen. In anotherform, the bispecific fusion antibody protein is divalent, consisting of,for example, an IgG with two binding sites for one antigen and two scFvwith two binding sites for a second antigen.

The invention further encompasses engineered antibodies with three ormore functional antigen-binding sites, including “Octopus antibodies”(see, e.g. US 2006/0025576A1), and “Dual Acting FAb” or “DAF” antibodiescomprising an antigen-binding site that binds to VISTA as well asanother, different antigen (see e.g. US 2008/0069820). Accordingly, thepresent invention includes bispecific molecules comprising at least onefirst binding specificity for VISTA and a second binding specificity fora second target epitope. According to at least some embodiments of theinvention, the second target epitope is an Fc receptor, e.g., humanFcyRI (CD64) or a human FcαR receptor (CD89). Therefore, the inventionincludes bispecific molecules capable of binding both to FcyR, FcαR orFcsR expressing effector cells (e.g., monocytes, macrophages orpolymorphonuclear cells (PMNs)), and to target cells expressing VISTA,respectively. These bispecific molecules target VISTA expressing cellsto effector cell and trigger Fc receptor-mediated effector cellactivities, such as phagocytosis of an VISTA expressing cells, antibodydependent cell-mediated cytotoxicity (ADCC), cytokine release, orgeneration of superoxide anion.

According to at least some embodiments of the invention in which thebispecific molecule is multispecific, the molecule can further include athird binding specificity, in addition to an anti-Fc bindingspecificity. In one embodiment, the third binding specificity is ananti-enhancement factor (EF) portion, e.g., a molecule which binds to asurface protein involved in cytotoxic activity and thereby increases theimmune response against the target cell.

The “anti-enhancement factor portion” can be an antibody, functionalantibody fragment or a ligand that binds to a given molecule, e.g., anantigen or a receptor, and thereby results in an enhancement of theeffect of the binding determinants for the Fc receptor or target cellantigen. The “anti-enhancement factor portion” can bind an Fc receptoror a target cell antigen. Alternatively, the anti-enhancement factorportion can bind to an entity that is different from the entity to whichthe first and second binding specificities bind. For example, theanti-enhancement factor portion can bind a cytotoxic T-cell (e.g., viaCD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell that resultsin an increased immune response against the target cell).

According to at least some embodiments of the invention, the bispecificmolecules comprise as a binding specificity at least one antibody, or anantibody fragment thereof, including, e.g., an Fab, Fab′, F(ab′)2, Fv,or a single chain Fv. The antibody may also be a light chain or heavychain dimer, or any minimal fragment thereof such as a Fv or a singlechain construct as described in Ladner et al. U.S. Pat. No. 4,946,778,the contents of which are expressly incorporated by reference.

In one embodiment, the binding specificity for an Fcy receptor isprovided by a monoclonal antibody, the binding of which is not blockedby human immunoglobulin G (IgG). As used herein, the term “IgG receptor”refers to any of the eight γ-chain genes located on chromosome 1. Thesegenes encode a total of twelve transmembrane or soluble receptorisoforms which are grouped into three Fcγ receptor classes: FcyRI(CD64), FcγRII (CD32), and FcγRIII (CD16). In one preferred embodiment,the Fcγ receptor is a human high affinity FcγRI. The human FcyRI is a 72kDa molecule, which shows high affinity for monomeric IgG. Theproduction and characterization of certain preferred anti-Fcγ monoclonalantibodies are described by Fanger et al. in PCT Publication WO 88/00052and in U.S. Pat. No. 4,954,617, the teachings of which are fullyincorporated by reference herein. These antibodies bind to an epitope ofFcγRI, FcγRII or FcγRIII at a site which is distinct from the Feybinding site of the receptor and, thus, their binding is not blockedsubstantially by physiological levels of IgG. Known anti-FcyRIantibodies include mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. Thehybridoma producing mAb 32 is available from the American Type CultureCollection, ATCC Accession No. HB9469. In other embodiments, theanti-Fcγ receptor antibody is a humanized form of monoclonal antibody 22(H22). The production and characterization of the H22 antibody isdescribed in Graziano, R. F. et al. (1995) J. Immunol. 155 (10):4996-5002 and PCT Publication WO 94/10332. The H22 antibody producingcell line is deposited at the American Type Culture Collection under thedesignation HA022CLI and has the accession no. CRL 11177.

In still other embodiments, the binding specificity for an Fc receptoris provided by an antibody that binds to a human IgA receptor, e.g., anFc-α receptor (Fc αRI(CD89)), the binding of which is preferably notblocked by human immunoglobulin A (IgA). The term “IgA receptor” isintended to include the gene product of one a-gene (FcαRI) located onchromosome 19. This gene is known to encode several alternativelyspliced transmembrane isoforms of 55 to 10 kDa. FcαRI (CD89) isconstitutively expressed on monocytes/macrophages, eosinophilic andneutrophilic granulocytes, but not on non-effector cell populations. FcαRI has medium affinity (Approximately 5×10⁻⁷ M⁻¹) for both IgA1 andIgA2, which is increased upon exposure to cytokines such as G-CSF orGM-CSF (Morton, H. C. et al. (1996) Critical Reviews in Immunology16:423-440). Four FcαRI-specific monoclonal antibodies, identified asA3, A59, A62 and A77, which bind FcαRI outside the IgA ligand bindingdomain, have been described (Monteiro, R. C. et al. (1992) J. Immunol.148: 1764).

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific molecules according to at least someembodiments of the invention are murine, chimeric and humanizedmonoclonal antibodies. The bispecific molecules of the present inventioncan be prepared by conjugating the constituent binding specificities,e.g., the anti-FcR and anti-VISTA binding specificities, using methodsknown in the art. For example, the binding specificity of eachbispecific molecule can be generated separately and then conjugated toone another. When the binding specificities are proteins or peptides, avariety of coupling or cross-linking agents can be used for covalentconjugation. Examples of cross-linking agents include protein A,carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyld-dithio propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160: 1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described in Paulus (1985) Behring Ins. Mitt. No.78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie etal. (1987)J. Immunol. 139: 2367-2375). Preferred conjugating agents areSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,II.). When the binding moieties are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAbXmAb, mAbXFab,FabXF(ab′)2 or ligandXFab fusion protein. A bispecific moleculeaccording to at least some embodiments of the invention can be a singlechain molecule comprising one single chain antibody and a bindingdeterminant, or a single chain bispecific molecule comprising twobinding determinants. Bispecific molecules may comprise at least twosingle chain molecules. Methods for preparing bispecific molecules aredescribed for example in U.S. Pat. No. 5,260,203; U.S. Pat. No.5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat. No. 5,132,405; U.S. Pat.No. 5,091,513; U.S. Pat. No. 5,476,786; U.S. Pat. No. 5,013,653; U.S.Pat. No. 5,258,498; and U.S. Pat. No. 5,482,858.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); controlled Fab-armexchange (see Labrijn et al., Proc. Natl. Acad. Sci. USA 110(13):5145-50(2013)); cross-linking two or more antibodies or fragments (see, e.g.,U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81 (1985));using leucine zippers to produce bi-specific antibodies (see, e.g.,Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992)); using “diabody”technology for making bispecific antibody fragments (see, e.g.,Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); andusing single-chain Fv (sFv) dimers (see, e.g. Gruber et al., J.Immunol., 152:5368 (1994)); and preparing trispecific antibodies asdescribed, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a γ counter or a scintillationcounter or by autoradiography.

Use of Agonistic Anti-Vista Antibodies and Pharmaceutical CompositionsContaining for Treatment of Autoimmune Disease

According to at least some embodiments, anti-VISTA antibodies,fragments, conjugates thereof or a pharmaceutical composition comprisingsame, as described herein, which function as VISTA stimulatingtherapeutic agents, may optionally be used for treating an immune systemrelated disease.

Optionally, the immune system related condition comprises an immunerelated condition, autoimmune diseases as recited herein, transplantrejection and graft versus host disease and/or for blocking or promotingimmune stimulation mediated by VISTA, immune related diseases as recitedherein and/or for immunotherapy (promoting or inhibiting immunestimulation).

Optionally the immune condition is selected from autoimmune disease,transplant rejection, inflammatory disease, allergic condition or graftversus host disease. Optionally the treatment is combined with anothermoiety useful for treating immune related condition.

Thus, treatment of multiple sclerosis using the agents according to atleast some embodiments of the present invention may be combined with,for example, any known therapeutic agent or method for treating multiplesclerosis, optionally as described herein.

Thus, treatment of rheumatoid arthritis or other arthritic condition,using the subject agonist antibodies may be combined with, for example,any known therapeutic agent or method for treating rheumatoid arthritis,optionally as described herein.

Thus, treatment of IBD, using the using the subject agonist antibodiesmay be combined with, for example, any known therapeutic agent or methodfor treating IBD, optionally as described herein.

Thus, treatment of psoriasis, using the subject agonist antibodies maybe combined with, for example, any known therapeutic agent or method fortreating psoriasis, optionally as described herein.

Thus, treatment of type 1 diabetes using the subject agonist antibodiesmay be combined with, for example, any known therapeutic agent or methodfor treating type 1 diabetes, optionally as described herein.

Thus, treatment of uveitis, using the subject agonist antibodies may becombined with, for example, any known therapeutic agent or method fortreating uveitis, optionally as described herein.

Thus, treatment of Sjögren's syndrome, using the subject agonistantibodies may be combined with, for example, any known therapeuticagent or method for treating for Sjögren's syndrome, optionally asdescribed herein.

Thus, treatment of systemic lupus erythematosus, using the subjectagonist antibodies may be combined with, for example, any knowntherapeutic agent or method for treating for systemic lupuserythematosus, optionally as described herein.

Thus, treatment of GVHD, using the subject agonist antibodies may becombined with, for example, any known therapeutic agent or method fortreating GVHD, optionally as described herein.

Thus, treatment of chronic or acute infection and/or hepatotoxicityassociated therewith, e.g., hepatitis, using the subject agonistantibodies may be combined with, for example, any known therapeuticagent or method for treating for chronic or acute infection and/orhepatotoxicity associated therewith, optionally as described herein.

In the above-described therapies preferably a subject with one of theaforementioned or other autoimmune or inflammatory conditions will beadministered an immmunoinhibitory anti-VISTA antibody disclosed hereinor antigen-binding fragment according to the invention, which antibodymimics or agonizes at least one VISTA-mediated effect on immunity, e.g.,it suppresses cytotoxic T cells, or NK activity and/or the production ofproinflammatory cytokines which are involved in the disease pathology,thereby preventing or ameliorating the disease symptoms and potentiallyresulting in prolonged disease remission, e.g., because of the inductionof Tregs which elicit T cell tolerance or prolonged immunosuppression.

The therapeutic agents and/or a pharmaceutical composition comprisingsame, as recited herein, according to at least some embodiments of theinvention, may be administered as the sole active ingredient or togetherwith other drugs in immunomodulating regimens or other anti-inflammatoryagents e.g. for the treatment or prevention of allo- or xenograft acuteor chronic rejection or inflammatory or autoimmune disorders, or toinduce tolerance.

Use of Agonistic Anti-Vista Antibodies and Pharmaceutical CompositionsContaining for Treatment of Sepsis

According to at least some embodiments, VISTA antibodies, fragments,conjugates thereof and/or pharmaceutical compositions as describedherein, may be used for treating sepsis. Sepsis is a potentiallylife-threatening complication of an infection. Sepsis represents acomplex clinical syndrome that develops when the initial host responseagainst an infection becomes inappropriately amplified and dysregulated,becoming harmful to the host. The initial hyperinflammatory phase(‘cytokine storm’) in sepsis is followed by a state of immunosuppression(Hotchkiss et al 2013 Lancet Infect. Dis. 13:260-268). This latter phaseof impaired immunity, also referred to as ‘immunoparalysis’, ismanifested in failure to clear the primary infection, reactivation ofviruses such as HSV and cytomegalovirus, and development of new,secondary infections, often with organisms that are not particularlyvirulent to the immunocompetent patient. The vast majority of septicpatients today survive their initial hyperinflammatory insult only toend up in the intensive care unit with sepsis-induced multiorgandysfunction over the ensuing days to weeks. Sepsis-inducedimmunosuppression is increasingly recognized as the overriding immunedysfunction in these vulnerable patients. The impaired pathogenclearance after primary infection and/or susceptibility to secondaryinfections contribute to the high rates of morbidity and mortalityassociated with sepsis.

According to at least some embodiments of the present invention, thereis provided use of a combination of the therapeutic agents and/or apharmaceutical composition comprising same, as recited herein, and aknown therapeutic agent effective for treating sepsis.

According to at least some embodiments of the present invention, thereis provided use of a combination of the therapeutic agents and/or apharmaceutical composition comprising same, as recited herein, can becombined with standard of care or novel treatments for sepsis, withtherapies that block the cytokine storm in the initial hyperinflammatoryphase of sepsis, and/or with therapies that have immunostimulatoryeffect in order to overcome the sepsis-induced immunosuppression phase.

Combination with standard of care treatments for sepsis, as recommendedby the “International Guidelines for Management of Severe Sepsis andSeptic Shock” (Dellinger et al 2013 Intensive Care Med 39: 165-228),some of which are described below.

-   -   1. Broad spectrum antibiotics having activity against all likely        pathogens (bacterial and/or fungal—treatment starts when sepsis        is diagnosed, but specific pathogen is not identified)—example        Cefotaxime (Claforan®), Ticarcillin and clavulanate (Timentin®),        Piperacillin and tazobactam (Zosyn®), Imipenem and cilastatin        (Primaxin®), Meropenem (Merrem®), Clindamycin (Cleocin),        Metronidazole (Flagyl®), Ceftriaxone (Rocephin®), Ciprofloxacin        (Cipro®), Cefepime (Maxipime®), Levofloxacin (Levaquin®),        Vancomycin or any combination of the listed drugs.    -   2. Vasopressors: example Norepinephrine, Dopamine, Epinephrine,        vasopressin    -   3. Steroids: example: Hydrocortisone, Dexamethasone, or        Fludrocortisone, intravenous or otherwise Inotropic therapy:        example Dobutamine for sepsis patients with myocardial        dysfunction    -   4. Recombinant human activated protein C (rhAPC), such as        drotrecogin alfa (activated) (DrotAA).    -   5. β-blockers additionally reduce local and systemic        inflammation. 6. Metabolic interventions such as pyruvate,        succinate or high dose insulin substitutions.

Use of Anti-Vista Antibodies and Pharmaceutical Compositions Containingfor Reducing the Undesirable Immune Activation that Follows Gene or CellTherapy or Transplant

As used herein the term “gene therapy” encompasses any type of genetherapy, vector-mediated gene therapy, gene transfer, virus-mediatedgene transfer and further encompasses certain cell therapies, e.g., CART and CAR NK cell therapies. According to at least some embodiments ofthe present invention, agonist VISTA antibodies, a fragment, a conjugatethereof and/or a pharmaceutical compositions as described herein, whichtarget VISTA and have inhibitory activity on immune responses, could beused as therapeutic agents for reducing the undesirable immuneactivation that follows gene or cell therapy used for treatment ofvarious genetic diseases. Without wishing to be limited by a singlehypothesis, such antibodies have VISTA-like inhibitory activity onimmune responses and/or enhance VISTA immune inhibitory activity,optionally by inhibition of pathogenic T cells and/or NK cells.

Many gene therapy products for the treatment of genetic diseases arecurrently in clinical trials. Recent studies document therapeuticsuccess for several genetic diseases using gene therapy vectors. Genetherapy strategies are characterized by 3 critical elements, the gene tobe transferred, the target tissue into which the gene will beintroduced, and the vector (gene delivery vehicle) used to facilitateentry of the gene to the target tissue. The vast majority of genetherapy clinical trials have exploited viral vectors as very efficientdelivery vehicles, including retroviruses, lentiviruses, adenoviruses,adeno-associated viruses, pseudotype viruses and herpes simplex viruses.However, the interactions between the human immune system and all thecomponents of gene therapy vectors seem to represent one of the majorlimitations to long-lasting therapeutic efficacy. Human studies haveshown that the likelihood of a host immune response to the viral vectoris high. Such immune responses to the virus or the transgene productitself, resulting in formation of neutralizing antibodies and/ordestruction of transduced cells by cytotoxic cells, can greatlyinterfere with therapeutic efficacy (Seregin and Amalfitano 2010 Viruses2:2013; Mingozzi and High 2013 Blood 122:23; Masat et al 2013 DiscovMed. 15:379). Therefore, developing strategies to circumvent immuneresponses and facilitate long-term expression of transgenic therapeuticproteins is one of the main challenges for the success of gene therapyin the clinic.

Factors influencing the immune response against transgenic proteinsencoded by viral vectors include route of administration, vector dose,immunogenicity of the transgenic protein, inflammatory status of thehost and capsid serotype. These factors are thought to influenceimmunogenicity by triggering innate immunity, cytokine production, APCmaturation, antigen presentation and, ultimately, priming of naive Tlymphocytes to functional effectors (Mingozzi and High 2013 Blood122:23). Therefore, the idea to dampen immune activation by interferingwith these very mechanisms has logically emerged with the aim to inducea short-term immunosuppression, avoid the early immune priming thatfollows vector administration and promote long-term tolerance.

As a strategy to inhibit the undesirable immune activation that followsgene therapy, particularly after multiple injections, immunomodulationtreatment by targeting of two non-redundant checkpoints of the immuneresponse at the time of vector delivery was tested in animal models.Studies of vector-mediated immune responses upon adenoviral vectorinstilled into the lung in mice or monkeys showed that transienttreatment with an anti-CD40L antibody lead to suppression ofadenovirus-induced immune responses; consequently, the animals could bere-administered with adenovirus vectors. Short treatment with this Abresulted in long-term effects on immune functions and prolongedinhibition of the adenovirus-specific humoral response well beyond thetime when the Ab effects were no longer significant, pointing to thetherapeutic potential in blockade of this costimulatory pathway as animmunomodulatory regimen to enable administration of gene transfervectors (Scaria et al. 1997 Gene Ther. 4: 611; Chirmule et al 2000 J.Virol. 74: 3345). Other studies showed that co-administration ofCTLA4-Ig and an anti-CD40L Ab around the time of primary vectoradministration decreased immune responses to the vector, prolonged longterm adenovirus-mediated gene expression and enabled secondaryadenovirus-mediated gene transfer even after the immunosuppressiveeffects of these agents were no longer present, indicating that it maybe possible to obtain persistence as well as secondaryadenoviral-mediated gene transfer with transient immunosuppressivetherapies (Kay et al 1997 Proc. Natl. Acad. Sci. U.S.A 94:4686). Inanother study, similar administration of CTLA4-Ig and an anti-CD40L Ababrogated the formation of neutralizing Abs against the vector, andenabled gene transfer expression, provided the treatment wasadministered during each gene transfer injection (Lorain et al 2008Molecular Therapy 16:541). Furthermore, administration of CTLA4-Ig tomice, even as single administration, resulted in suppression of immuneresponses and prolonged transgene expression at early time points(Adriouch et al 2011 Front. Microbiol. 2: 199). However, CTLA4-Ig alonewas not sufficient to permanently wipe out the immune responses againstthe transgene product. Combined treatment targeting two immunecheckpoints with CTLA4-Ig and PD-L1 or PDL-2 resulted in synergisticimprovement of transgene tolerance at later time points, by probablytargeting two non-redundant mechanisms of immunomodulation, resulting inlong term transgene persistence and expression (Adriouch et al 2011Front. Microbiol. 2: 199).

According to at least some embodiments of the present invention, thesubject agonists may be used to overcome the limitation of immuneresponses to gene therapy, could be used for reducing the undesirableimmune activation that follows gene therapy alone or with other actives.Current approaches include exclusion of patients with antibodies to thedelivery vector, administration of high vector doses, use of emptycapsids to adsorb anti-vector antibodies allowing for subsequent vectortransduction, repeated plasma exchange (plasmapheresis) cycles to adsorbimmunoglobulins and reduce the anti-vector antibody titer.

Novel approaches attempting to overcome these limitations can be dividedinto two broad categories: selective modification of the Ad vectoritself and pre-emptive immune modulation of the host (Seregin andAmalfitano 2010 Viruses 2:2013). The first category comprises severalinnovative strategies including: (1) Ad-capsid-display of specificinhibitors or ligands; (2) covalent modifications of the entire Advector capsid moiety; (3) the use of tissue specific promoters and localadministration routes; (4) the use of genome modified Ads; and (5) thedevelopment of chimeric or alternative serotype Ads.

The second category of methods includes the use of immunosuppressivedrugs or specific compounds to block important immune pathways, whichare known to be induced by viral vectors. Immunosuppressive agents havebeen tested in preclinical studies and shown efficacy in prevention oreradication of immune responses to the transfer vector and transgeneproduct. These include general immunosuppressive agents such ascyclosporine A; cyclophosphamide; FK506; glucocorticoids or steroidssuch as dexamethasone; TLR9 blockade such as the TLR9 antagonistoligonucleotide ODN-2088; TNF-a blockade with anti-TNF-a antibodies orTNFR-Ig antibody, Erk and other signaling inhibitors such as U0126. Inthe clinical setting, administration of glucocorticoids has beensuccessfully used to blunt T cell responses directed against the viralcapsid upon liver gene transfer of adenovirus-associated virus (AAV)vector expressing human factor IX transgene to severe hemophilia Bpatients (Nathwani et al 2011 N. Engl. J. Med. 365:2357).

In contrast to the previous approaches that utilize drugs that tend to“globally” and non-specifically immunosuppress the host, more selectiveimmunosuppressive approaches have been developed. These include the useof agents which provide blockade of positive co-stimulatoryinteractions, such as between CD40 and CD154, ICOS and ICOSL, CD28 andCD80 or CD86 (including CTLA4-Ig), NKG2D and NKG2D ligands, LFA-1 andICAM, LFA-3 and CD2, 4-1BB and 4-1BBL, OX40 and OX40L, GITR and GITRLand agents that stimulate negative costimulatory receptors such asCTLA-4, PD-1, BTLA, LAG-3, TIM-1, TEVI-3, KIRs, and the receptors forB7-H4 and B7-H3. Some of these have been utilized in preclinical orclinical transplantation studies (Pilat et al 2011 Sem. Immunol.23:293).

In the above-described gene or cell therapies or in treating transplantindications preferably a subject who has or is to receive cell or genetherapy or a transplanted tissue or organ will be administered animmmunoinhibitory anti-VISTA antibody disclosed herein orantigen-binding fragment according to the invention, which antibodyenhances, agonizes or mimics at least one VISTA-mediated effect onimmunity, e.g., its inhibitory effect on cytotoxic T cells or NKactivity and/or its inhibitory effect on the production ofproinflammatory cytokines, or its stimulatory effect on Tregs therebypreventing or reducing host immune responses against the cell or geneused in therapy or an undesired immune response against the transplantedcells, organ or tissue. Preferably the treatment will elicit prolongedimmune tolerance against the transplanted or infused cells, tissue ororgan. In some instances, e.g., in the case of transplanted cells,tissues or organs containing immune cells, the immmunoinhibitoryanti-VISTA antibody disclosed herein or antigen-binding fragment may becontacted with the cells, tissue or organ prior to infusion ortransplant, and/or potentially immune cells of the transplant recipientin order to tolerize the immune cells and potentially prevent anundesired immune response or GVHD immune reaction.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination ofanti-human VISTA antibodies according to the invention and optionallyanother immunosuppressive or other active agent. Thus, the presentinvention features a pharmaceutical composition comprising atherapeutically effective amount of anti-human VISTA antibodiesaccording to at least some embodiments of the present invention. Inparticular the present invention features a pharmaceutical compositioncomprising a therapeutically effective [immunosuppressive]amount of atleast one agonist anti-human VISTA antibody or antibody fragmentaccording to the present invention.

A pharmaceutical composition according to at least some embodiments ofthe present invention may be used for the treatment of immune relateddisorders, autoimmunity, allergy, GVHD, inflammation or hepatotoxicityassociated with infectious disorder and/or sepsis. “Treatment” refers toboth therapeutic treatment and prophylactic or preventative measures.Those in need of treatment include those already with the disorder aswell as those in which the disorder is to be prevented. Hence, themammal to be treated herein may have been diagnosed as having thedisorder or may be predisposed or susceptible to the disorder. “Mammal”for purposes of treatment refers to any animal classified as a mammal,including humans, domestic and farm animals, and zoo, sports, or petanimals, such as dogs, horses, cats, cows, etc. Preferably, the mammalis human.

The term “therapeutically effective amount” refers to an amount of agentaccording to the present invention that is effective to treat a diseaseor disorder in a mammal. The therapeutic agents of the present inventioncan be provided to the subject alone or as part of a pharmaceuticalcomposition where they are mixed with a pharmaceutically acceptablecarrier. In many instances agonist or antagonist anti-VISTA antibodiesaccording to the invention will be used in combination with otherimmunotherapeutics or other therapeutic agents useful in treating aspecific condition.

A composition is said to be a “pharmaceutically acceptable carrier” ifits administration can be tolerated by a recipient patient. As usedherein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion).

Such compositions include sterile water, buffered saline (e.g.,Tris-HCl, acetate, phosphate), pH and ionic strength and optionallyadditives such as detergents and solubilizing agents (e.g., Polysorbate20, Polysorbate 80), antioxidants (e.g., ascorbic acid, sodiummetabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol). Non-aqueous solvents orvehicles may also be used as detailed below.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions according to at least someembodiments of the invention include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants. Depending on the route ofadministration, the active compound, i.e., monoclonal or polyclonalantibodies and antigen-binding fragments and conjugates containing same,and/or alternative scaffolds, that specifically bind any one of VISTAproteins, or bispecific molecule, may be coated in a material to protectthe compound from the action of acids and other natural conditions thatmay inactivate the compound. The pharmaceutical compounds according toat least some embodiments of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66: 1-19).Examples of such salts include acid addition salts and base additionsalts. Acid addition salts include those derived from nontoxic inorganicacids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,hydriodic, phosphorous and the like, as well as from nontoxic organicacids such as aliphatic mono- and dicarboxylic acids, phenyl-substitutedalkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic andaromatic sulfonic acids and the like. Base addition salts include thosederived from alkaline earth metals, such as sodium, potassium,magnesium, calcium and the like, as well as from nontoxic organicamines, such as N,N′-dibenzylethylenediamine, N-methylglucamine,chloroprocaine, choline, diethanolamine, ethylenediamine, procaine andthe like.

A pharmaceutical composition according to at least some embodiments ofthe invention also may include a pharmaceutically acceptableanti-oxidant. Examples of pharmaceutically acceptable antioxidantsinclude: (1) water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, a-tocopherol, and the like; and (3) metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositionsaccording to at least some embodiments of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin. Sterile injectable solutionscan be prepared by incorporating the active compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

A composition of the present invention can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for therapeutic agentsaccording to at least some embodiments of the invention includeintravascular delivery (e.g. injection or infusion), intravenous,intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, oral,enteral, rectal, pulmonary (e.g. inhalation), nasal, topical (includingtransdermal, buccal and sublingual), intravesical, intravitreal,intraperitoneal, vaginal, brain delivery (e.g. intra-cerebroventricular,intracerebral, and convection enhanced diffusion), CNS delivery (e.g.intrathecal, perispinal, and intra-spinal) or parenteral (includingsubcutaneous, intramuscular, intravenous and intradermal), transmucosal(e.g., sublingual administration), administration or administration viaan implant, or other parenteral routes of administration, for example byinjection or infusion, or other delivery routes and/or forms ofadministration known in the art. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion. In a specific embodiment, a protein, a therapeutic agent or apharmaceutical composition according to at least some embodiments of thepresent invention can be administered intraperitoneally orintravenously.

Alternatively, a VISTA specific antibody according to the invention canbe administered via a non-parenteral route, such as a topical, epidermalor mucosal route of administration, for example, intranasally, orally,vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition according to at least some embodiments of the invention canbe administered with a needles hypodermic injection device, such as thedevices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335;5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-knownimplants and modules useful in the present invention include: U.S. Pat.No. 4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate; U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering medicamentsthrough the skin; U.S. Pat. No. 4,447,233, which discloses a medicationinfusion pump for delivering medication at a precise infusion rate; U.S.Pat. No. 4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. These patents are incorporated herein byreference. Many other such implants, delivery systems, and modules areknown to those skilled in the art.

In certain embodiments, the anti-VISTA antibodies can be formulated toensure proper distribution in vivo. For example, the blood-brain barrier(BBB) excludes many highly hydrophilic compounds. To ensure that thetherapeutic compounds according to at least some embodiments of theinvention cross the BBB (if desired), they can be formulated, forexample, in liposomes. For methods of manufacturing liposomes, see,e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomesmay comprise one or more moieties which are selectively transported intospecific cells or organs, thus enhance targeted drug delivery (see,e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplarytargeting moieties include folate or biotin (see, e.g., U.S. Pat. No.5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem.Biophys. Res. Commun. 153: 1038); antibodies (P. G. Bloeman et al.(1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. AgentsChemother. 39: 180); surfactant protein A receptor (Briscoe et al.(1995) Am. J Physiol. 1233: 134); pI20 (Schreier et al. (1994) J. Biol.Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.346: 123; J. J. Killion; and I. J. Fidler (1994) Immunomethods 4:273.

In yet another embodiment, immunoconjugates of the invention can be usedto target compounds (e.g., therapeutic agents, labels, cytotoxins,radiotoxins immunosuppressants, etc.) to cells which have VISTA cellsurface receptors by linking such compounds to the antibody disclosedherein. Thus, the invention also provides methods for localizing ex vivoor in vivo cells expressing VISTA (e.g., with a detectable label, suchas a radioisotope, a fluorescent compound, an enzyme, or an enzymeco-factor). Alternatively, the immunoconjugates can be used to killcells which have VISTA cell surface receptors by targeting cytotoxins orradiotoxins to VISTA antigen.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., solublepolypeptide conjugate containing the ectodomain of the VISTA antigen,antibody, immunoconjugate, alternative scaffolds, and/or bispecificmolecule, may be coated in a material to protect the compound from theaction of acids and other natural conditions that may inactivate thecompound. The pharmaceutical compounds according to at least someembodiments of the present invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66: 1-19).Examples of such salts include acid addition salts and base additionsalts. Acid addition salts include those derived from nontoxic inorganicacids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,hydroiodic, phosphorous and the like, as well as from nontoxic organicacids such as aliphatic mono- and dicarboxylic acids, phenyl-substitutedalkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic andaromatic sulfonic acids and the like. Base addition salts include thosederived from alkaline earth metals, such as sodium, potassium,magnesium, calcium and the like, as well as from nontoxic organicamines, such as N,N′-dibenzylethylenediamine, N-methylglucamine,chloroprocaine, choline, diethanolamine, ethylenediamine, procaine andthe like.

A pharmaceutical composition according to at least some embodiments ofthe present invention also may include a pharmaceutically acceptableanti-oxidant. Examples of pharmaceutically acceptable antioxidantsinclude: (1) water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, a-tocopherol, and the like; and (3) metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Examplesof suitable aqueous and nonaqueous carriers that may be employed in thepharmaceutical compositions according to at least some embodiments ofthe present invention include water, ethanol, polyols (such as glycerol,propylene glycol, polyethylene glycol, and the like), and suitablemixtures thereof, vegetable oils, such as olive oil, and injectableorganic esters, such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of coating materials, such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositionsaccording to at least some embodiments of the present invention iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin. Sterile injectable solutionscan be prepared by incorporating the active compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about I percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms according to at least some embodiments of thepresent invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

For administration of the VISTA antibody disclosed herein, the dosageranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg,of the host body weight. For example dosages can be 0.3 mg/kg bodyweight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplarytreatment regime entails administration once per week, once every twoweeks, once every three weeks, once every four weeks, once a month, onceevery 3 months or once every three to 6 months. Preferred dosageregimens for an antibody disclosed herein according to at least someembodiments of the present invention include 1 mg/kg body weight or 3mg/kg body weight via intravenous administration, with the antibodydisclosed herein being given using one of the following dosingschedules: (i) every four weeks for six dosages, then every threemonths; (ii) every three weeks; (iii) 3 mg/kg body weight once followedby 1 mg/kg body weight every three weeks.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously; in which case thedosage of each antibody disclosed herein administered falls within theranges indicated. Antibody disclosed herein is usually administered onmultiple occasions. Intervals between single dosages can be, forexample, daily, weekly, monthly, every three months or yearly. Intervalscan also be irregular as indicated by measuring blood levels of antibodyto the target antigen in the patient. In some methods, dosage isadjusted to achieve a plasma antibody concentration of about 1-1000mug/ml and in some methods about 25-300 microgram/ml.

Alternatively, therapeutic agent can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of thetherapeutic agent in the patient. In general, human antibodies show thelongest half-life, followed by humanized antibodies, chimericantibodies, and nonhuman antibodies. The half-life for fusion proteinsmay vary widely. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

Functional Screening Using In Vivo Animal Models

1. Testing of Vista Agonist Antibodies According to the Invention inConcanavalin A-Induced Hepatitis Animal Model.

Autoimmune hepatitis (AIH) is a chronic inflammatory disease of theliver, characterized by the loss of self-tolerance leading to B and Tcell responses against the liver. The ConA model represents thebest-characterized system for understanding the pathogenesis of AIH.ConA is a lectin that binds to specific sugar moieties, which areenriched in the liver. The modification of these sugar residues by ConAresults in rapid CD4⁺ T cell activation through interaction withmodified MHC structures expressed by liver macrophages. An intense, buttransient, cytokine production occurs with most canonical T cellcytokines (IL-2, IL-3, IFNγ and TNFα) reaching peak plasma levels within4-6 hours. Notably, ConA induced inflammation can be blocked bydepleting CD4+ T cells. The ConA model with hV-KI mice may be used toconfirm suppressive activity of agonistic anti-VISTA mAbs according tothe invention. Mice are weighed and treated with 10 mpk of anti-VISTAantibody or the appropriate isotype control 3 hours prior to injectionwith 15 mpk of ConA. The anti-VISTA mAbs are administered I.P. whileConA is injected via the tail-vein in these mice. At the 6-hourtime-point post ConA administration, the mice are euthanized and bloodis collected. The plasma fraction is then be analyzed for plasmacytokines by a multiplex assay for 32 cytokines. Each antibody is testedtwo times in independent experiments to confirm activity. For eachcytokine in the 32-plex, a one-way ANOVA will be performed, with aDunnett's post-test to compare each anti-VISTA antibody to the isotypecontrol. The tested anti-VISTA mAb is ranked based upon efficacy ofcytokine suppression (how much was the cytokine suppressed) andvariability (how consistent is the suppression within each experimentand between experiments). Additional emphasis is placed on mAb thatsuppress cytokines that are canonically associated with T cellactivation.

Numerous anti-human VISTA antibodies according to the invention havebeen screened in the ConA model and were efficacious (immunosuppressive)therein, i.e., they suppressed ConA-induced cytokine production andpromoted survival and in particular suppressed the expression ofcytokines involved in T cell activation including IL-2. Particularly,the inventors tested INX800, INX801, and INX903 as well as agonistanti-murine VISTA antibodies and all were efficacious(immunosuppressive) in the ConA hepatitis model. Therefore, agonistanti-human VISTA antibodies according to the invention should be usefulin treating/preventing inflammation and hepatotoxicity associated withsome chronic and acute infectious conditions such as hepatitis.

2. Testing of Vista Agonist Antibodies According to the Invention inGraft Versus Host Disease Animal Models

GVHD is a systemic disease mediated by adoptive transfer of allogeneic Tcells into an irradiated host. There are five major steps that arecritical in the pathogenesis of GVHD; 1) Damage to the host, mostcommonly in the form of the irradiation event that precedes the T celltransfer; 2) Activation of the allogeneic T cells by both host and donorAPCs; 3) Expansion of the T cells in the lymph nodes and spleen; 4)Trafficking into peripheral sites such as the skin, gut, liver and lung;and 5) Damage to the host driven by T cells and also recruited myeloidcells. In certain models, such as F1→Parental strain, a chronic GVHDoccurs that is a suitable model for lupus as the mice developanti-nuclear mAb and immune complex mediated glomerular nephritis. Ofnote, genetic deletion of VISTA from the donor T cells results in a moreaggressive form of GHVD than seen in mice receiving WT T cells.

This assay may be used to identify and rank agonism of agonisticanti-human VISTA candidates. Also this assay may be used to confirm thatagonist antibodies according to the invention may be used to treat orprevent GVHD. In this model BALB/c mice are lethally irradiated andgiven allogeneic bone marrow and splenic T cells from hV-KI mice toinduce GVHD; with one group not receiving T cells as a negative control.Mice receiving the allogeneic T cells are split into the control Iggroup and the treatment groups. Up to four unique VISTA mAb will be usedin a single experiment, with eight mice per group, and two replicateexperiments will be conducted. 10 mpk or another dose of antibody isadministered at the time of T cell transfer, as well as at days 2 and 4post transfer. The body weight of each mouse will be tracked, and anymouse that loses more than 20% of its initial starting bodyweight willbe sacrificed. Kaplan Meier curves are generated for each experimentwith a log-rank statistical test comparing each anti-VISTA antibody tothe control. Should all four VISTA mAb fully protect against GVHD, thendose response assays will be run in the GVHD model with groups beingtreated with 10, 3, 1 and 0.3 mpk of antibody. LD50 values will becalculated for each antibody.

A number of agonist anti-human VISTA antibodies according to theinvention were evaluated in this animal model. These tested antibodiesall were efficacious (immunosuppressive) in this model, i.e., theyreduced the symptoms of the disease, slowed disease progression, reduceddisease-associated weight loss and promoted survival. Particularly, eachof INX800, INX801, INX901, INX902, INX903 and INX904 were evaluated andwere demonstrated to alleviated or prevent disease symptoms in thisanimal model. Also, it was determined using the A and B forms of INX901that either the A or B form were equally effective in the GVHD animalmodel.

3. Testing of Vista Agonist Antibodies According to the Invention in anAnimal Model of Inflammatory Bowel Disease.

Inflammatory bowel diseases (IBD), Crohn's disease and ulcerativecolitis result from incompletely defined and complex interactionsbetween host immune responses, genetic susceptibility, environmentalfactors, and the enteric luminal contents. Recent genome-wideassociation studies report associations between immune cell regulatorygenes and IBD susceptibility. Both innate and adaptive immune cellintrinsic genes are represented in these studies, indicating a centralrole for these cell populations in IBD pathogenesis. There currentlyexist more than 50 animal models of human IBD. While no one modelperfectly phenocopies human IBD, many are useful for studying variousaspects of human disease, including disease onset and progression andthe wound-healing response.

In one well established IBD model intestinal inflammation is initiatedwith syngeneic splenic CD4⁺ CD45RB T cell adoptive transfer into T and Bcell deficient recipient mice. The CD4+ CD45RBhi T cell populationcontains mainly naive T cells primed for activation that are capable ofinducing chronic small bowel and colonic inflammation. This methodallows the researcher to modify key experimental variables, includingboth innate and adaptive immune cell populations, to answer biologicallyrelevant questions relating to disease pathogenesis. Additionally, thismethod provides precise initiation of disease onset and awell-characterized experimental time course permitting the kinetic studyof clinical features of disease progression in mice. Intestinalinflammation induced by this method shares many features with human IBD,including chronic large and small bowel transmural inflammation,pathogenesis driven by cytokines such as TNF and IL-12, and systemicsymptoms such as wasting. Thus, it is an ideal model system for studyingthe pathogenesis of human IBD.

An agonistic anti-human VISTA antibody according to the invention(INX901) was tested and shown to be efficacious in this IBD model.Particularly this agonist antibody was demonstrated to suppress cytokinelevels and to effectively prevent or inhibit (i) colitis related weightloss, (ii) weight loss associated with colitis progression, (iii) colonshortening, (iv) the recruitment of inflammatory infiltrates to thecolon and (v) the development of colitis. Therefore, agonist VISTAantibodies according to the invention may be used in the treatment ofIBD and related inflammatory and intestinal conditions.

4. Testing of Vista Agonist Antibodies According to the Invention inLupus Animal Models.

Lupus is an autoimmune or inflammatory condition with symptoms includingkidney inflammation, increased proteinuria, and splenomegaly. There are4 types of lupus of which Systemic Lupus Erythematosus or (“SLE”) is themost common form. This disease can be mild or severe and can affectmajor organ systems. Lupus is an autoimmune condition of unknown causethat may result in inflammation of the kidneys—called lupusnephritis—which can affect the body's ability to filter waste from theblood, and or if severe may result in kidney damage requiring dialysisor kidney transplant. Also SLE may result in an increase in bloodpressure in the lungs—called pulmonary hypertension—which can causedifficulty breathing. Further SLE may cause Inflammation of the nervoussystem and brain which can cause memory problems, confusion, headaches,and strokes. Further SLE may result in inflammation in the brain's bloodvessels which can cause high fevers, seizures, and behavioral changes.Also SLE may result in hardening of the arteries or coronary arterydisease—the buildup of deposits on coronary artery walls—can lead to aheart attack.

Agonistic anti-human VISTA antibodies according to the invention(INX903, INX901, INX901-A and INX901-B) and anti-murine VISTA antibodieswere tested and shown to be efficacious in different lupus modelsincluding the MRL/Ipr lupus model, the NZBWF-1 lupus model and the B6D2Fmodel. The B6D2F model is a murine model wherein SLE is induced by thetransfer of human VISTA knock-in DDE1 CD8 depleted splenocytes (donor)into a B6D2F1 host (recipient) In this model, donor CD4 T cellpolyclonal activation drives cognate host B cell activation, expansion,and their production of autoantibodies leading to renal disease.Lupus-like features of B6 CD8 depleted transferred to B6D2F1 modelinclude: (1) Immune complex glomerulonephritis; (2) anti-nuclear abs;(3) anti-dsDNA abs; and (4) anti-RBC abs (Coombs positivity).Additionally, this model meets sex-based differences in renal diseaseseverity.

In these 3 different lupus models agonistic anti-human and murine VISTAantibodies were demonstrated to be efficacious and to reduce theincidence of lupus disease development, disease progression, reduceproteinuria levels, inhibit nephritis and kidney damage, reduce T cellactivation and accumulation, reduce B cell activation and accumulation,and to inhibit autoantibody production. Particularly, INX903, INX901,INX901-A and INX901-B were shown to (i) reduce T cell proliferation andactivation, (ii) reduce cognate B cell activation (MHCII expression) andaccumulation, reduce splenomegaly, reduce anti-dsDNA IgG autoantibodyproduction and to reduce type I interferon signature. Also theseimmunosuppressive effects were not impacted by whether the human IgG2constant region of the antibody was in the A or B form. Therefore,agonist VISTA antibodies according to the invention may be used in thetreatment of lupus and related inflammatory and autoimmune conditions.

5. Testing of Vista Agonist Antibodies in A Psoriasis Animal ModelImiquimod (IMQD) Induced Psoriasis Model

The ability of anti-VISTA antibodies to treat psoriasis was evaluatedusing the Imiquimod (IMQD) induced Psoriasis Model. Imiquimod (IMQD) isa commercially available cream containing TLR7/8 agonists that is widelyused for dermatological conditions such as viral infections andmelanoma. Application of IMQD to the skin over multiple days results inthickening of the epidermis via proliferation of the keratinocytes.Additionally, an immunological infiltration into the dermis layeroccurs, with populations of both T cells and myeloid cells. Recurrentadministration of IMQD creates a skin lesion similar to what is observedin patients with Psoriasis. IL-17 and IL-23 are thought to be the majorcytokines involved in the immune response to IMQD.

An agonistic anti-murine VISTA antibody was tested and shown to beefficacious in this psoriasis model. Particularly, this antibody reducedthe number of CD3⁺ T cells infiltrating Imiquimod treated skin. Based onthe observed results VISTA agonist antibodies may be used in thetreatment or prevention of psoriasis and other T cell mediatedautoimmune or inflammatory skin conditions.

6. Testing of Vista Agonist Antibodies in Arthritis Animal Model

The immunosuppressive effects of anti-VISTA antibodies to treatarthritis may be tested in different animal models. Agonisticanti-murine and anti-human VISTA antibodies were tested and shown to beefficacious in a well-accepted arthritis model, i.e., the Collageninduced arthritis or CIA Model. INX800, INX901, INX902 and INX903 aswell as a hamster anti-murine anti-VISTA antibodies were all tested inthis arthritis model. Disease development was assessed by measuringinflammation swelling in the affected joints over time. Clinical scoringwas accomplished by awarding a score of 1 for each swollen digit, ascore of 5 for a swollen footpad and a score of 5 for a swollen wrist orankle (Charles River Labs scoring system), which added together give amaximal score of 60 for each animal.

As described infra each of these antibodies decreased the arthritisdisease and INX901 and INX902 significantly decreased disease scope.Based on these results anti-human VISTA agonist antibodies may be usedin the treatment or prevention of rheumatoid arthritis and otherinflammatory or autoimmune conditions.

Having described the invention the following examples are provided tofurther illustrate the invention and its inherent advantages.

Examples Example 1: Use of Assays to Screen for ImmunosuppressiveAnti-Mouse VISTA Abs

The present inventors developed various assays to screen for putativeagonistic anti-mouse VISTA antibodies. As shown in FIG. 1 in vitro andin vivo screening assays were used to identify immunosuppressiveanti-VISTA mAbs. In the experiments in FIG. 1A purified T cells wereplated on top of anti-CD3 in the presence of the indicated mAb for 72hours. Proliferation was measured by H3 incorporation. In theexperiments in FIG. 1B purified DO11.10 T cells were stimulated by ISQpulsed APCs for 6 days in the presence of the indicated antibody.Proliferation was measured through use of CTV dilution dye. In theexperiments in FIG. 1C GVHD was induced by transfer of C57BL/6 cellsinto irradiated BALB/c recipients. Mice were injected I.P. with 200 μgof antibody on day 0, 2 and 4 post transfer and survival was analyzed.In the experiments in FIG. 1D mice were treated with 10 mpk of theindicated antibody 3 hours prior to administration of ConA (15 mpk) andIL-2 was analyzed in plasma at 6 by Luminex.

More particularly, in the first assay, CD4⁺ T cells were isolated andincubated with Ab1, Ab2 or Ab3 before being added to anti-CD3 coatedplates. After 3 days in culture, the T cells were pulsed with tritiatedthymidine, which is incorporated by proliferating cells. Notably, bothAb1 and Ab2 induced a significant reduction in the proliferative rate ofthe T cells, while Ab3 had no effect (FIG. 1) In a similar assay wheretransgenic T cells were stimulated with antigen pulsed APCs instead, Tcell activation was measured by proliferative dye dilution. Similar tothe anti-CD3 assay, Ab1 suppressed antigen-specific T cell proliferationby ˜50% (FIG. 1B). These data indicate that the Ab3 mAb blocks mVISTAfunction (i.e., enhances immune responses) whereas Ab1 and Ab3 stimulatemVISTA function and down regulate key immune responses.

We also determined whether Ab3 and Ab1 could be distinguished using invivo animal models, particularly in GVHD and ConA hepatitis models. Micewith GVHD which were treated with a control antibody (Ham Ig) hadprogressive disease and needed to be euthanized by 4 weeks post graft asexpected (FIG. 1C). Ab3 treated mice were also susceptible to GVHD, andin fact most mice died prior to the control treated group, indicatingAb3 may exacerbate disease. Conversely, all of the Ab1 treated miceshowed no obvious symptoms of GVHD and almost all were healthy for atleast 40 days. Specifically in these experiments mice with GVHD treatedwith a control antibody (Ham Ig) had progressive disease and needed tobe euthanized by 4 weeks post graft as expected (FIG. 1C). Ab3 treatedmice were also susceptible to GVHD, and in fact most mice died prior tothe control treated group, indicating Ab3 may exacerbate disease.Conversely, all of the Ab1 treated mice showed no obvious symptoms ofGVHD and almost all were healthy for at least 40 days.

In the ConA model, the inventors tested whether each VISTA antibodywould impact the well-characterized T cell cytokine response to ConA.Notably Ab1, but not Ab3, induced decreased plasma cytokine levels ofIL-2 (FIG. 1D). Specifically, in the ConA model, the inventors furthertested whether each VISTA antibody would impact the well-characterized Tcell cytokine response to ConA. Notably Ab1, but not Ab3, induceddecreased plasma cytokine levels of IL-2 (FIG. 1D).

Accordingly these results demonstrate that both anti-VISTA mAbs (Ab1 andAb2) are immunosuppressive and it has also been shown that suchimmunosuppressive anti-mouse VISTA antibodies can be distinguished frominflammatory immunosuppressive anti-mouse VISTA antibodies (Ab3). Asshown in FIG. 1 Ab1 is efficacious (immunosuppressive) in multipleinflammatory models including GVHD, NZB/W F1 lupus-likeglomerulonephritis, concanavalin A (ConA)-induced hepatitis, collagenantibody induced arthritis (CAIA), and Imiquimod induced psoriasis. Ineach of these diseases, administration of Ab1 during the progression ofdisease greatly diminished pathology and/or mortality. Each model listedhas a unique requirement on T cells for disease progression. GVHD andConA are both driven by Th1 T cell responses.

Example 2: Identification of Anti-VISTA Abs which SuppressesAutoimmunity in Different Autoimmune Disease Models

In the experiments in FIG. 2A-F the effects of different anti-mouseVISTA Abs were again compared in different disease models. In theexperiments in FIG. 2A NZB/W F1 mice were treated 3×/week with eitherAb1 or Ham Ig (200 μg) starting at 25 weeks until the end of theexperiment. “×” denotes time points where the control treated group hadall been sacrificed. In the experiment in FIG. 2B mice were treated with200 μg of antibody 3 hours prior to administration of 15 mg/kg (mpk) ofConA and survival was followed for 80 hours. In the experiment in FIG.2C mice were treated sequentially with Collagen II mAb followed by LPSand arthritis was measured by measuring for paw swelling. In theexperiments Ab1 and Ham-Ig were administered (200 μg) 3× every otherday. In the experiment in FIG. 2D Imiquimod was applied to the ear ofmice daily. At day 14, Ab1 or Ham-Ig (200 μg) were administered everyother day and ear thickness was measured with calipers. In theexperiment in the same FIG. 2E-F imiquimod was applied to the backs ofmice daily. At day 9, mice were euthanized and skin was sectioned &stained for CD3 expression by IHC.

As shown in FIG. 2A-F, in each of these experimental models,administration of Ab1 during the progression of the particular diseasegreatly diminished pathology and/or mortality. Each model listed has aunique requirement on T cells for disease progression. GVHD and ConA areboth driven by Th1 T cell responses.

Imiquimod induced psoriasis is an IL-17/23 driven disease where T cellsare recruited into the dermal layer of the skin. Ab1 drastically reducedthe number of CD3⁺ cells in the dermis (FIGS. 2E and F), but had noimpact on splenic T cell populations (data not shown), indicating thatthis anti-mouse VISTA Ab preferentially suppressed immunity at theinflammatory lesion.

NZB/W F1 lupus is a multifactorial disease with contributions from Bcells, T cells and myeloid cells. In this model, therapeuticadministration of Ab1 reduced proteinuria levels indicating decreaseddamage to the kidneys. Finally, CAIA does not involve adaptive immunity,instead being driven by macrophages and granulocytes. Suppression byanti-VISTA in this model indicates that the antibody may also impactupon the myeloid compartment. As such, suppressive VISTA mAb appear tomediate effects on both the T cell and innate immune compartments.

Therefore, as shown in FIG. 1 and FIG. 2 both monoclonal hamsteranti-mouse VISTA Abs Ab1 and AB2 induced a significant reduction in theproliferative rate of the T cells, while Ab3 had no effect (FIG. 1). Ina similar assay where transgenic T cells were stimulated with antigenpulsed APCs, T cell activation was measured by proliferative dyedilution. Similar to the anti-CD3 assay, Ab1 suppressed antigen-specificT cell proliferation by ˜50% (FIG. 1B). These data suggest that Ab1 andAb2 stimulate VISTA function and thereby down regulate key immuneresponses.

Particularly, Ab1, a hamster anti-mouse VISTA antibody was efficaciousin multiple inflammatory models including GVHD, NZB/W F1 lupus-likeglomerulonephritis, concanavalin A (ConA)-induced hepatitis, collagenantibody induced arthritis (CAIA), and Imiquimod induced psoriasis(FIGS. 1 and 2). In each of these diseases, administration of Ab1 duringthe progression of disease greatly diminished pathology and/ormortality. Each model listed has a unique requirement on T cells fordisease progression. GVHD and ConA are both driven by Th1 T cellresponses. As noted above, Imiquimod induced psoriasis is an IL-17/23driven disease where T cells are recruited into the dermal layer of theskin. Therefore, suppression by Ab1 in this particular autoimmune modelindicates that this antibody may also be affecting the myeloidcompartment. Therefore, these immunosuppressive anti-mouse VISTA mAb'sappear to mediate effects on both the T cell and innate immunecompartments.

Example 3: Development of Human VISTA Knock-in Mice for Use in Screeningfor Agonistic Anti-Human VISTA Abs

The previous examples relate to the isolation and characterization ofagonistic anti-mouse VISTA Abs. Heretofore an agonistic anti-human VISTAAb has never been reported in the literature. This is despite the factthat very many antagonistic anti-human VISTA antibodies have beenidentified by the present Assignee and other groups. Accordingly, priorto this invention it was uncertain whether agonistic anti-human VISTAantibodies would be identified.

Such antibodies would be highly beneficial as currently there is noapproved human therapeutics that exploit the natural function of NCR'sto suppress the immune response. Although Orencia (CTLA4-Ig) iseffective, it only acts by blocking the CD28-B7 interaction and pathwayand does not work by stimulating a downregulatory pathway. Asillustrated by the potent immunosuppressive effects of 2 differentagonistic anti-VISTA mAbs as shown in the examples which follow, theengagement of this pathway may prove to be a revolution in themanagement of different human autoimmune diseases. Moreover, theimmunosuppressive impact of anti-VISTA on both adaptive and innateautoimmune effector mechanisms sets it apart from many otheranti-inflammatory agents.

With respect to the foregoing, it was hypothesized that a desirable andnecessary reagent in screening for agonistic anti-human VISTA Abs is ahuman VISTA knock-in mouse. A human VISTA knock-in mouse has beencreated by the present Assignee (“hV-KI Mouse”). These hV-KI miceexpress human VISTA in replacement of mouse VISTA. Particularly, asshown in FIG. 3 CD4⁺ T cells, CD8⁺ T cells, Tregs (CD4⁺ FoxP3⁺), andmonocytes, CD11b⁺, Ly6C⁺, Ly6G⁻ were isolated from the lymph nodes of WTand VISTA KI mice, and stained with αVISTA antibodies against mouse orhuman protein respectively. The expression pattern of the hV-KI isidentical to what is seen in WT mice as CD4⁺ and CD8⁺ T cells,regulatory T cells and monocytes all express consistent amounts ofsurface protein between the two strains (see FIG. 3).

Additionally, hV-KI mice do not develop any signs of inflammatorydisease that are observed in VISTA KO mice, indicating that hVISTA isfully functional within the mouse immune system (data not shown).Accordingly, this mouse model may be used in different assays to screenfor immunosuppressive mAbs.

Example 4: Synthesis of Putative Agonistic Anti-Human VISTA Antibodies

The sequences of different anti-human VISTA antibodies is contained inFIG. 4. These antibodies specifically bind to human VISTA, e.g.,VSTB49-VSTB116, and possess VISTA antagonist properties, i.e., theseantibodies inhibit the suppressive effects of VISTA on immunity when inthe IgG1 format, e.g., when the antibody comprises an IgG1 Fc regionwhich is wild-type, i.e., unmodified.

Among the antibodies identified in FIG. 4 is 1E8. This murine anti-humanVISTA antibody comprises the variable heavy and light chain polypeptidesset forth below and was converted by the inventors into two humanchimeric forms. The first chimeric antibody referred to herein as INX800was obtained by the attachment of human IgG2 heavy and light constantregion polypeptides to the 1E8 variable heavy and light chainpolypeptides. In this first chimeric antibody none of the amino acidresidues within the IgG2 constant regions were modified.

The second chimeric antibody referred to herein as INX801 was similarlyobtained by the attachment of human IgG2 heavy and light constant regionpolypeptides to the 1E8 variable heavy and light chain polypeptides. Inthis second chimeric antibody the cysteine residue at position 127within the human IgG2 kappa chain was converted into a serine. Otherwisenone of the amino acid residues within the IgG2 constant regions weremodified.

1E8 V_(H) Polypeptide (SEQ ID NO: 57)EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEVYPDSSTINYTPSLKDKFIISRDNAKNTLYLQMIKVRSEDTALYYCARGR GDYWGQGTSVTVSS1E8 V_(L) Polypeptide (SEQ ID NO: 58)DIQMTQSPASLSASVGETVTITCRASGNIHNYLSWYHQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQNFWSTPFTFGS GTKLEIKR.

Example 5: Evaluation of Putative Agonistic Anti-Human VISTA Antibodiesin ConA Animal Model

The effects of both chimeric IgG2 antibodies and control antibodies werecompared in a Concavalin A Hepatitis model. In this in vivo modeldifferent animals were predosed with 10 mg/kg of either chimeric IgG2antibody (INX800 or INX801) or with a control antibody 3 hours prior toConcavalin A administration. 3 hours after antibody administration themice were then dosed with ConA at 12 mg/kg. These animals and thecontrols were then bled by cardiac puncture 6 hours after ConA dosing.All of the mice appeared fine, no obvious morbidity or mortality.

The blood was then analyzed for cytokine expression. Particularly, a32-plex was run using plasma obtained from the collected blood samplesusing conventional methods and cytokine test kit conventionally used forcytokine analysis. As shown in FIG. 5 the expression of severalproinflammatory cytokines was significantly suppressed in the animalsadministered INX800 or INX801 antibodies compared to the controlanimals. Particularly, GM-CSF, IL-2, IL-4, IL-6, IL-17 and TNF-α levelswere all significantly lower in the INX800 or INX801 treated animalscompared to the controls. [Reduced] expression of these cytokines wassubstantially identical in the INX800 or INX801 treated animals.

Also, the expression of certain chemokines (keratinocyte derivedchemokine or “KC”) and macrophage inflammatory protein 2 (MIP-2) weresubstantially increased in the INX800 or INX801 treated animals comparedto the controls. Again, the [increased] expression of these proteins wassubstantially identical in the INX800 or INX801 treated animals. Basedon these results both INX800 and INX801 appear to be potent VISTAagonists as they appear to elicit the analogous immunosuppressiveeffects that VISTA elicits ion the expression of various inflammatorycytokines.

Example 6: Evaluation of Putative Agonistic Anti-Human VISTA Antibodiesin Graft Versus Host Disease (GVHD) Animal Model

The effects of the same putative agonistic anti-human VISTA antibodies,INX800 and INX801 were also compared in a graft versus host disease(GVHD) animal model compared to untreated animals or controls treatedwith irrelevant antibody. In this animal model T cells were adoptivelytransferred into irradiated hosts and body-weight was measured as a readout of disease. Based on GVHD disease progression all of the Controlmice (8/8) had to be euthanized. The results of these animal studies areshown in FIG. 6. As shown none of the INX800 or INX801 [0/8] treatedmice needed to be euthanized as GVHD was considerably depressed as aresult of treatment with INX800 or INX801 antibody. Based on theseresults both INX800 and INX801 appear to be potent VISTA agonists asthey appear to potently suppress GVHD immune responses.

Example 7: Effects of Putative Agonistic Anti-Human VISTA Antibodies onCD3-Driven T Cell Immune Responses

The effects of the same agonistic anti-human VISTA antibodies, INX800and INX801 were also compared as to their potential to suppressCD3-driven T cell immune response. In these experiments plates werecoated with OKT3 (2.5 μg/ml). T cells were the preincubated withantibody for 30 minutes. The antibody treated T cells were then added tothe OKT3 coated plates and the T cells cultured on these plates for 72hours. As a readout of the possible effects of the antibodies onCD3-driven T cell immune responses T cell proliferation was determinedusing Tritium incorporation methods, a well-accepted method fordetecting T cell proliferation. As shown in FIG. 7, T cell proliferationwas considerably reduced in the cultured T cells which were treated withINX800 or INX801 antibodies compared to the control T cell cultures.

Example 8: Effects of Putative Agonistic Anti-Human VISTA Antibodies onSpecific T Cell Populations and Total T Cell Numbers

Experiments were also affected in order to compare the possible effectsof the same anti-human VISTA antibodies, INX800 and INX801, on thenumbers of specific T cells as well as on the total number of T cells.These experiments were conducted in order to assess whether the observedeffects of the subject anti-human VISTA antibodies on cytokines and Tcells could have been attributable to cell depletion (a non-specificeffect) rather than the antibodies eliciting an immunosuppressive effectbased on their promoting specific VISTA-mediated immunosuppressiveeffects on immunity.

Both agonistic anti-human VISTA antibodies, INX800 and INX801, had nosignificant effect on the number of specific T cell populations, or onthe total number of T cells. Moreover, the results with both the INX800and INX801 antibodies were substantially the same. The results ofexemplary experiments are in FIG. 8.

Based thereon, the observed agonistic effects of INX800 and INX801 donot appear to be attributable to cell depletion. Rather, both of theseantibodies appear to elicit an immunosuppressive effect on T cellactivation/proliferation, GVHD immune responses and the expression ofproinflammatory cytokines based on their promoting specificVISTA-mediated immunosuppressive effects on immunity.

Example 9: Summary of Effects of Different Agonistic Anti-Human VISTAAbs in Different Immune Models

As shown in Table 1 and 2 below the agonistic or immunosuppressiveeffects of different anti-human VISTA antibodies was evaluated havingthe sequences are in FIG. 4. To date 12 different chimeric anti-humanVISTA antibodies have been demonstrated to be immunosuppressive. Some ofthe results obtained to date are summarized in the Tables. Antibodies inBin 1 all compete for binding to human VISTA but do not compete forVISTA binding with antibodies in Bin 2. Conversely, the anti-human VISTAantibodies in Bin 2 all compete for binding to human VISTA with eachother but not with antibodies in Bin 1.

The antibody in Table 2 which is marked “inconclusive” eliciteddifferent effects, including immunosuppressive effects in the same assayor elicited ambiguous results for other reasons. As shown in Table 1 and2 a total of 12 anti-human VISTA antibodies have been isolated which areimmunosuppressive in MLR assays or ConA assays and/or other in vitro andin vivo assays or autoimmune, inflammatory or GVHD disease models andwhich mimic or agonize the immunosuppressive effects of human VISTA.Based on these results it is expected that other anti-human VISTAantibodies may be obtained by analogous methods including those havingthe same or different VISTA epitopic specificity.

Also, the experiments in FIG. 9 compare the effects of differentanti-human VISTA antibodies in ConA assays and on the expression ofselect proinflammatory cytokines and inflammation markers, I.e., IL-2, γInterferon and IL-12p70.

TABLE 1 (HUMAN OR HUMANIZED ANTI-HUMAN VISTA ANTIBODIES) 1st SuppressionAssay as IgG2 2nd MLR In MLR Assay Prolif and/or MLR Epitope as ConA HepProlif. as mAb ID Group Origin IgG1 Kd, M Status Assay IgG1)INX903|VSTB95 1 HFA ++ 1.26E− Tested + Hybr 10 For (His) immuno-suppression INX904|VSTB103 1 Phage, − 6.36E− Tested +/− yes original 10For immuno- suppression INX905|VSTB53 1 HFA ++ 2.64E− Tested ++ Hybr(Fc) 11 For immuno- suppression INX908|VSTB92 1 HFA ++ 9.34E− **Tested++ Hybr (Fc) 11 For immuno- suppression INX900|VSTB50 2 HFA ++ 6.32E−Tested +/− Hybr (Fc) 10 For immuno- suppression INX901|VSTB56 2 HFA +/−2.35E− Tested ++ yes Hybr (Fc) 11 For immuno- suppression INX902|VSTB632 HFA +/− 8.30E− Tested ++ yes Hybr (Fc) 10 For immuno- suppressionINX906|VSTB54 2 HFA +/− 2.53E− Tested ++ Hybr (Fc) 11 For immuno-suppression INX907|VSTB66 2 HFA +/− 8.06E− Tested + yes Hybr (Fc) 11 Forimmuno- suppression INX909|VSTB67 1 HFA +/− 6.29E− To be Hybr (Fc) 11tested INX913|VSTB85 1 HFA ++ 3.78E− To be Hybr 11 tested (InterFA D)INX914|VSTB97 1 Phage, +/− 7.68E− To be original 10 testedINX915|VSTB106 1 Phage, +/− 1.67E− To be ILM 10 tested INX916|VSTB107 1Phage, ++ 8.90E− To be ILM 11 tested INX917|VSTB110 1 Phage, +/− 2.02E−To be ILM 10 tested INX918|VSTB113 1 Phage, ++ 4.33E− To be ILM 11tested INX919|VSTB115 1 Phage, +/− 1.45E− To be yes ILM 10 testedINX910|VSTB73 2 HFA +/− 2.26E− To be yes Hybr 09 tested (His)INX911|VSTB76 2 HFA +/− 1.31E− To be Hybr 09 tested (His) INX912|VSTB842 HFA + 2.03E− To be Hybr 09 tested (InterFA D) VSTB100 1 Phage, +/−1.48E− original 09 VSTB101 1 Phage, +/− 3.18E− original 09 VSTB102 1Phage, +/− 2.98E− original 09 VSTB104 1 Phage, + 6.75E− original 10VSTB105 1 Phage, + 1.15E− ILM 10 VSTB108 1 Phage, + 4.94E− ILM 10VSTB109 1 Phage, +/− 1.02E− ILM 10 VSTB111 1 Phage, ++ 1.71E− ILM 10VSTB112 1 Phage, ++ 1.56E− ILM 10 VSTB114 1 Phage, ++ 1.52E− ILM 10VSTB116 1 Phage, ++ 2.13E− ILM 10 VSTB49 1 HFA + 5.07E− Hybr (Fc) 10VSTB51 1 HFA ++ 1.04E− Hybr (Fc) 10 VSTB59 1 HFA + 1.06E− Hybr (Fc) 10VSTB65 1 HFA ++ 1.08E− Hybr (Fc) 09 VSTB70 1 HFA +/− 2.23E− Hybr 09(His) VSTB81 1 HFA +/− 3.12E− Hybr 10 (InterFA D) VSTB98 1 Phage, +2.28E− original 09 VSTB99 1 Phage, +/− 1.54E− original 09 VSTB60 2 HFA +3.56E− Hybr (Fc) 10 VSTB78 2 HFA ++ 1.13E− Hybr 09 (InterFA D) VSTB74 4HFA − 5.62E− Hybr 10 (His)

TABLE 2 (MURINE ANTI-HUMAN VISTA ANTIBODIES) Antibody Bin Suppressive?MLR Prolif. Kd, M 1E8* 1 Yes ++ NT GG8 1 Yes ++ NT GA1 2 Inconclusive −NT *Shown to be immunosuppressive in 2 different IgG2 forms.

Example 10: Determination of Epitopes Of Anti-Human VISTA Antibodies ByB Cell Epitope Mapping

The epitopic specificity of some putative agonistic anti-human VISTAantibodies was determined using custom peptide arrays using fragments ofhuman VISTA, using proprietary methods [ProArray Ultra™] Essentially,the determination of peptide-antibody binding was performed byincubation of antibody samples with a ProArray Ultra™ peptidemicroarray, followed by incubation with a fluorescently labeledsecondary antibody. After several washing steps the ProArray Ultra™arrays were dried and scanned using a high-resolution fluorescencemicroarray scanning.

All peptides (listed below) are synthesized separately, and then boundto the ProArray Ultra™ slide surface using ProImmune's proprietarytechnology. This optimized process ensures that peptides are presentedon the array in such a manner as to closely mimic the properties of thecorresponding protein region, circumventing the inherent physiochemicalvariation of the free peptides themselves and making a compatible,combined peptide and protein array platform. The test analytes (peptidesand proteins) are dispensed onto the ProArray Ultra™ slide in discretespots and appropriate gal-files enable exact alignment of the resultingarray features back to the analyte deposited.

Peptide-antibody binding is determined by incubation of antibody samples(provided by the customer) with the ProArray Ultra™ slides, followed byincubation with a fluorescently labeled secondary antibody. After thefinal incubation and washing steps the microarrays are dried and scannedin a high-resolution microarray scanning system.

After scanning the fluorescently labeled ProArray Ultra™ slides, thescanner records an image which is evaluated using image analysissoftware—enabling interpretation and quantification of the levels offluorescent intensities associated with each fluorescent spot on thescanned microarray slide. The peptide microarray was based on anoverlapping peptide library synthesized from the human VISTA polypeptidesequence. Based on the sequence 15-mer microarray peptides, overlappingby 12 amino acids, were generated using ProImmune's ProArray Ultra™technology. Details of the peptides synthesized are listed in TABLE 3(below). ‘Position’ refers to the start and end amino acid within thepolypeptide sequence from which the peptide was derived. Synthesizedpeptides were immobilised onto ProArray Ultra™ slides in 24 identicalsub-arrays, each comprising test-peptides and control features insextuplicate spots. The peptides are shown in Table 3 below.

TABLE 3 ProArray Ultra ™ Peptide Details Peptide ID Position Sequence  1 1-15 FKVATPYSLY VCPEG (SEQ ID NO: 7)  2  4-18 ATPYSLYVCP EGQNV (SEQID NO: 8)  3  7-21 YSLYVCPEGQ NVTLT (SEQ ID NO: 9)  4 10-24 YVCPEGQNVTLTCRL (SEQ ID NO: 10)  5 13-27 PEGQNVTLTC RLLGP (SEQ ID NO: 11)  616-30 QNVTLTCRLL GPVDK (SEQ ID NO: 12)  7 19-33 TLTCRLLGPV DKGHD (SEQID NO: 13)  8 22-36 CRLLGPVDKG HDVTF (SKI ID NO: 14)  9 25-39 LGPVDKGHDVTFYKT (SEQ ID NO: 15) 10 28-42 VDKGHDVTF YKTWYR (SEQ ID NO: 16) 1131-45 GHDVTFYKT WYRSSR (SEQ ID NO: 17) 12 34-48 VTFYKTWYRS SRGEV (SEQ IDNO: 18) 13 37-51 YKTWYRSSRG EVQTC (SEQ ID NO: 19) 14 40-54 WYRSSRGEVQTCSER (SEQ ID NO: 20) 15 43-57 SSRGEVQTCS ERRPI (SEQ ID NO: 21) 1646-60 GEVQTCSERR PIRNL (SEQ ID NO: 22) 17 49-63 QTCSERRPIR NLTFQ (SEQ IDNO: 23) 18 52-66 SERRPIRNLTF ODLH (SEQ ID NO: 24) 19 55-69 RPIRNLTFQDLHLHH (SEQ ID NO: 25) 20 58-72 RNLTFQIDLHL HHGGH (SEQ ID NO: 26) 2161-75 TFQDLHLHH GGHQAA (SEQ ID NO: 27) 22 64-78 DLHLHHGGH QAANTS (SEQ IDNO: 28) 23 67-81 LHHGGHQAA NTSHDL (SEQ ID NO: 29) 24 70-84 GGHQAANTSHDLAQR (SEQ ID NO: 30) 25 73-87 QAANTSHDL AQRHGL (SEQ ID NO: 31) 2676-90 NTSHDLAQR FIGLESA (SEQ ID NO: 32) 27 79-93 HDLAQRHGL ESASDH(SEQ ID NO: 33) 28 82-96 AQRHGLESAS DHFIGN (SEQ ID NO: 34) 29 85-99HGLESASDH FIGNFSI (SEQ ID NO: 35) 30  88-102 ESASDHHGN FSITMR (SEQ IDNO: 36) 31  91-105 SDHHGNESIT MRNLT (SEQ ID NO: 37) 32  94-108FIGNESITMR NLTLLD SEQ ID NO: 38) 33  97-111 FSITMRNLTLL DSGL (SEQ IDNO: 39) 34 100-114 TMRNLILLIDS GLYCC (SEQ ID NO: 40) 35 103-117NLTLLDSGLY CCLVV (SEQ ID NO: 41) 36 106-120 LLIDSGLYCCLV VEIR (SEQ IDNO: 42) 37 109-123 SGLYCCLVVEI RHHH (SEQ ID NO: 43) 38 112-126YCCLVVEIRH HHSEH (SEQ ID NO: 44) 39 115-129 LVVEIRHHHS EHRVH (SEQ IDNO: 45) 40 118-132 EIRHHHSEHR VHGAM (SEQ ID NO: 46) 41 121-135 HHH5EHRVHGAMELQ (SEQ ID NO: 47) 42 124-138 SEHRVHGAM ELQVQT (SEQ ID NO: 48) 43127-141 RVHGAMELQ VQTGKD (SEQ ID NO: 49) 44 130-144 GAMELQVQT GKDAPS(SEQ ID NO: 50) 45 133-147 ELQVQTGKD AP5NCV (SEQ ID NO: 51) 46 136-150VQTGKDAPS NCVVYP (SEQ ID NO: 52) 47 139-153 GKDAPSNCV VYPSSS (SEQ IDNO: 53) 48 142-156 APSNCVVYPS SSQDS (SEQ ID NO: 54) 49 145-159NCVVYPSSSQ DSENI (SEQ ID NO: 55) 50 148-162 VYPSSSQDSE NITAA (SEQ IDNO: 56)

The results of this epitope analysis with particular anti-human VISTAantibodies are summarized in FIG. 4 and in the examples below.

Example 11: Epitope Binning Assay

Additionally the epitopic binding properties of some anti-human VISTAantibodies having sequences shown in FIG. 4 were characterized byplacing these antibodies into different epitope “bins” based on theirbinding characteristics as described below.

Methods: ProteOn XPR36 system (BioRad) was used to perform epitopebinning. ProteOn GLC chips (BioRad, Cat#176-5011) were coated with twosets of 6 monoclonal antibodies (mAbs) using the manufacturerinstructions for amine-coupling chemistry (BioRad, cat #176-2410).Competing mAbs were pre-incubated in excess (250 nM final concentration)with human VISTA (25 nM final concentration) for 4 hours at roomtemperature and 6 at a time were run over the chip coated with thepanels of coated mAbs with an association time of 4 minutes followed bydissociation for 5 minutes. Following each run, the chips wereregenerated with 100 nM phosphoric acid.

The data analysis involved grouping all sensorgrams by ligand andapplying an alignment wizard, which automatically performs an X and Yaxis alignment, and artifact removal. An Interspot correction was thenapplied to the data.

A non-competing mAb was defined as having a binding signal the sameor >Al signal (binding to human VISTA only). A competing mAb was definedas having binding signal <<Al signal (i.e., binding to human VISTAonly). For example VSTB49 and VSTB51 complexed with VISTA did not bindto the VSTB85 coated on the chip and therefore were classified ascompeting for the same binding site on VISTA as VSTB85. The results ofthis binning analysis with particular anti-human VISTA antibodies aresummarized in FIG. 4.

Example 12: Epitope Mapping of Anti-VISTA Antibodies UsingHydrogen/Deuterium (H D) Exchange Studies

Antibody epitopes of anti-VISTA antibodies may be identified by variousmethods such as alanine scanning and Hydrogen/Deuterium (H D) Exchangeand overlapping peptide arrays as described in the previous Example.Another exemplary means for identifying epitopes of putative agonisticanti-human VISTA antibodies is described below.

To identify the epitopes for VSTB50, 60, 95 and 112 on human VISTA,solution hydrogen/deuterium exchange-mass spectrometry (HDX-MS) wasperformed using the corresponding Fabs. For H/D exchange, the proceduresused to analyze the Fab perturbation were similar to that describedpreviously (Hamuro et al, J. Biomol. Techniques 14:171-182, 2003; Hornet al, Biochemistry 45:8488-8498, 2006) with some modifications. Fabswere prepared from the IgGs with papain digestion and Protein A captureusing Pierce Fab Preparation Kit (Thermo Scientific, Cat#44985). Thehuman VISTA protein sequence contains six N-linked glycosylation sites.To improve the sequence coverage, the protein was deglycosylated withPNGase F. The deglycosylated VISTA protein was incubated in a deuteratedwater solution for predetermined times resulting in deuteriumincorporation at exchangeable hydrogen atoms. The deuterated VISTAprotein was in complex with a Fab of VSTB50, VSTB60, VSTB95 or VSTB112in 46 deuterium oxide (D20) at 4° C. for 30 sec, 2 min, 10 min and 60min. The exchange reaction was quenched by low pH and the proteins weredigested with pepsin. The deuterium levels at the identified peptideswere monitored from the mass shift on LC-MS. As a reference control,VISTA protein was processed similarly except that it was not in complexwith the Fab molecules. Regions bound to the Fab were inferred to bethose sites relatively protected from exchange and, thus, containing ahigher fraction of deuterium than the reference VISTA protein. About 94%of the protein could be mapped to specific peptides.

The solution HDX-MS perturbation maps of VISTA with VSTB50/VSTB60, andVSTB95/VSTB112 were mapped and two epitope groups were identified.Anti-VISTA VSTB50 recognizes the same epitope as VSTB60 does; VSTB95binds to another epitope region as VSTB112 does on VISTA. Anti-VISTAVSTB50 and 60 share the same epitope which comprises segments, 103NLTLLDSGL111 (SEQ ID NO:59), and 136VQTGKDAPSNC146 (SEQ ID NO:60)Anti-VISTA VSTB95 and VSTB112 appear to target similar epitopes,comprising segments 27PVDKGHDVTF36 (SEQ ID NO:61), and 54RRPIRDLTFQDL65(SEQ ID NO:62). These HDX-MS results provide the peptide level epitopesfor exemplary anti-VISTA antibodies having the sequences identified inFIG. 4. There were no overlapping epitope regions for these two epitopegroups. These results are in agreement with the previous competitionbinning data in that they do not compete with each other. Again theepitope analysis results for various anti-human VISTA antibodiesanalyzed as described herein is summarized in FIG. 4.

Example 13: Evaluation of the Role of the Human IgG2 Backbone on α-HumanVISTA Antibody INX901 Agonist/Immune-Suppressive Activity in DifferentIn Vitro and In Vivo Models

Antibodies on a native human IgG2 backbone exist as a mixture ofisoforms caused by disulfide bond shuffling among cysteines present inthe heavy chain hinge, CH1, and light chain (Zhang, A., (2015),“Conformational difference in human IgG2 disulfide isoforms revealed byhydrogen/deuterium exchange mass spectrometry”, Biochemistry, 54(10),1956-1962; FIG. 10). These isoforms were assessed by RP-HPLC (FIG. 10),based on methods developed by Dillon et al., “Optimization of areversed-phase high-performance liquid chromatography/mass spectrometrymethod for characterizing recombinant antibody heterogeneity andstability”, J Chromatography A, 1120(1), 112-120. The optimized methodused a shallower and higher organic mobile phase B content relative tothat in Dillon (id). Separate A and B forms enriched from INX901 wereprepared closely following the conditions reported in Dillon (id) butcombined with a buffer exchange back into DPBS and an endotoxin removalprocedure employed subsequent to the enrichment reactions (FIG. 11).

In the course of preparing these experiments it was observed thatreversion of the A-enriched form occurs more quickly than expected, andat lower residual redox reagent concentrations than expected.Utilization of a fast-spin, size-exclusion based desalting procedure wastherefore employed, which appeared to largely prevent this reversion. Asshown in panel (A) in FIG. 10 disulfide shuffling leads to isoforms Aand B, along with the transition for A/B (reproduced from Zhang, A. etal., 2015). (B) Isoforms are distinguishable by RP-HPLC (figure fromZhang, A. et al., 2015). (C) Observed RP-HPLC chromatogram for INX901.

The inventors optimized RP-HPLC Method for detecting IgG2 isoforms isdescribed below. In FIG. 11: (Black line, top) the chromatogram shows adominant left-most peak defining the B-form. (Red line, bottom)Chromatogram shows a dominant right peak defining the A-form.

Optimized RP-HPLC Methods for Isoform Detection

Mobile Phase A Preparation (0.1% v/v TFA in water):

-   -   1. Measured 1.0 L Milli-Q water in a 1.0 L graduated cylinder    -   2. Added 1.0 mL of TFA to the 1 L of water using a 1 mL glass        Hamilton syringe    -   3. Transferred the solution to a 1 L bottle, mixed well.    -   4. Expiry is 2 weeks after preparation        Mobile Phase B Preparation (70% v/v IPA, 20% v/v ACN, 9.9% v/v        water, 0.1% v/v TFA):    -   1. Measured 700 mL IPA into a 1.0 L graduated cylinder    -   2. Measured 200 mL ACN into a 250 mL graduated cylinder and        transferred to the 1.0 L graduated cylinder containing the 700        mL IPA    -   3. Added Milli-Q water to the 1.0 L graduated cylinder        containing the 700 mL IPA and 200 mL ACN until the liquid        reached to 1.0 L mark    -   4. Added 1.0 mL of TFA to the 1 L of water using a 1 mL glass        Hamilton syringe    -   5. Transferred the solution to a 1 L bottle, mixed well.    -   6. Expiry is 2 weeks after preparation

RP-HPLC Chromatography Conditions

-   -   1. Column A (large bore): Zorbax 300SB-C8, 5 μm, 2.1×150 mm,        <<OR>>    -   2. Column B (narrow bore): Zorbax 300SB-C8, 3.5 μm, 1×50 mm    -   3. Mobile Phase A: 0.1% v/v TFA in water    -   4. Mobile Phase B: 70% v/v IPA, 20% v/v ACN, 9.9% v/v water,        0.1% v/v TFA    -   5. Flow rate: 0.5 mL/min for Column A or 0.25 mL/min for Column        B    -   6. Column compartment: 75.0±1.0° C.    -   7. Detection: 214 nm    -   8. RP-HPLC mobile phase gradient (Table below)

Time (min) Mobile Phase B % 0 15 2 26 34 36 35 75 36 15 40 15

INX901 Disulfide Isoform Enrichment Methods

B-Form Enrichment

-   -   1. Into endotoxin free non-pyrogenic tube, add:        -   2.1 mL of INX901 (5.66 mg/mL)        -   792.6 μL 1 M Tris pH 8.0        -   495.4 μL endo-free water        -   396.3 additional endo-free water        -   237.8 μL of 100 mM Cysteine        -   39.6 μL of 100 mM Cystamine    -   2. Finger vortex (lightly), then place capped at 2-8° C. for 24        hr    -   3. Soaked Pall microsep spin-concentrator in 0.3M NaOH 2 hr at        RT, then rinsed 3× with 10×DPBS, then 3× with endo-free water.        Air dried in BSC before use    -   4. Followed vendor's instructions for regenerating 0.5 mL        endotoxin removal column, using the 0.2N NaOH/95% ethanol (2 hrs        at RT) option for step 3; used 1×DPBS as final equilibration        buffer    -   5. Concentrated ˜4,020 μL of reaction (from Step 2) in a        separate PALL microsep (as prepared above).    -   6. Concentrated at 2,500×G for 35 min to less 0.4 mL (≧10×) then        re-diluted with 4 mL 1× DPBS, repeated 2 additional times    -   7. Concentrated at 2,500×G for 15 min to below 2 mL, then added        back 1×DPBS to 2 mL    -   8. Added all 2 mL of buffer exchanged sample to the regenerated,        spun dried, bottom capped endotoxin removal column, capped the        top tightly, inverted, placed at room temp-inverted 3 more times        every ˜20 minutes, then spun out the sample into non-pyrogenic        tube (1 min at 500×G, as per Vendor's instructions), placed at        2-8° C.

A-Form Enrichment

-   -   1. Into endotoxin free non-pyrogenic tube, add:        -   1750 μL INX901 (6.2 mg/mL)        -   370 μL endo-free water        -   700 μL 1M Tris pH8.0        -   435 μL 8M GdCl        -   210 μL 0.1 M Cysteine HCl (made fresh from 1 M stock)        -   35 μL 0.1 M Cystamine-2HCl (made fresh from 1 M stock)        -   (Final volume 3500 μL)    -   2. Finger vortex (lightly), then place capped at 2-8® C. for 24        hr    -   3. Prepared #7-2 mL Zeba spin columns (Thermo P/N 89890) as per        vendor's instructions, equilibrating into 1× Dulbecco's        Phosphate Buffered Saline (DPBS).    -   4. Loaded 500 μL of the above reaction mixture onto each of the        #7, and spun 2 minutes at 1000×G (also as per vendor's        instructions), collecting into clean pyrogen free tubes.    -   5. Placed in de-pyrogenated PALL microsep, spun total of 1 hour,        10 minutes, concentrated to approximately 1.7 mL at        approximately 5 mg/mL    -   6. Added all 1.7 mL above to one 0.5 mL endotoxin removal spin        column (Thermo P/N 88274) prepared as per Vendor's instructions        (including overnight in 0.2 M NaOH at room tempo), equilibrated        into 1×DPBS. Left at room temp approximately 1 hr, then placed        at 4° C. for approximately another 1 hr, in both cases inverting        the capped tube about every 15 minutes.    -   7. Recovered prep by spinning 500×G for 1 minute (also as per        vendor's instructions).    -   8. Recovered volume: approximately 1.3 mL at 4.61 mg/mL (all        concentrations based on the NanoDrop's built-in IgG extinction        coefficient of 0.73)

IgG2 A- and B-Locked Variants

Specific substitutions to the amino acid sequence of IgG2 are capable ofpreventing disulfide shuffling, and depending on the mutation willresult in a locked conformation that is either A-like or B-like(Martinez, et al., (2008). “Disulfide connectivity of humanimmunoglobulin G2 structural isoforms”, Biochemistry, 47(28), 7496-7508;Allen, et al., (2009), “Interchain disulfide bonding in human IgG2antibodies probed by site-directed mutagenesis”, Biochemistry, 48(17),3755-3766.

The inventors therefore designed INX901 and INX908 variants with eitherthe C233S (A-locked) or C127S (B-locked) mutation (Eu numbering) tomatch the IgG2 variants used by White et al., (2015), “Conformation ofthe human immunoglobulin G2 hinge imparts superagonistic properties toimmunostimulatory anticancer antibodies”, Cancer Cell, 27(1), 138-148.

Constant heavy chain sequences are listed below.

IgG2 C2335 (A-locked) (SEQ ID NO: 63)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVIVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVNNAKTKPREEQFNSTFRVVSVLIVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPCK IgG2 C1275 (B-locked) (SEQ ID NO: 64)ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVIVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVNNAKTKPREEQFNSTFRVVSVLIVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPCK

Silent Fc Variants

The inventors designed INX901 and INX908 variants with a silent Fcregion by introducing the following point mutations on an IgG1 backbone:L234A/L235A/G237A/P238A/H268A/A330S/P331S (McCarthy et al., (2015) U.S.patent application Ser. No. 14/818,864. Washington, D.C.: U.S. In onetype of variant (INX901Si and INX908Si), the CH1/hinge region of theheavy constant region is native IgG1, which does not support thedisulfide shuffling of a native IgG2 (FIG. 12, middle). In a second typeof variant (INX901HSi and INX908HSi), the CH1/hinge region is nativeIgG2, which does support disulfide shuffling (White, A. L. et al., 2015)(FIG. 12, bottom). Constant heavy chain sequences for both types ofvariants are listed below

IgG1 with silent Fc (INX901Si and INX908Si) (SEQ ID NO: 65)ASTKGPSVFPLAPSSKSTSGGTAALGCLVICYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVIVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHICPPCPAPEAAGASSVFLFFTKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgG2 CH1/hinge +IgG1 silent Fc (INX901HSi and INX908HSi) (SEQ ID NO: 66)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVIVPS5NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPEAAGASSVFLFPPKPKOTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYRSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The experiments in FIG. 12 compare the immune properties of INX901Fc-silent variants with respect to disulfide shuffling. (Top) INX901 onan IgG2 backbone exhibits an expected mixture of A, A/B, and B isoforms.(Middle) INX901Si on a silent IgG1 backbone exists as a single isoform.(Bottom) INX901HSi possesses an IgG1 silent Fc region with a CH1/hingefrom IgG2, which enables disulfide shuffling equivalent to native IgG2.These results indicate that FcR binding appears to affect the agonistproperties of the inventive antibodies.

Example 14: Function of INX901 and INX908 in Various Ig Backbones toDetermine Requirement of Hinge and Fc Regions

We conducted experiments to assess the functional requirements of theCH1/hinge and Fc regions of the heavy chain of the anti-human VISTAantibodies, INX901 and INX908. In their original state, both moleculesare on native human IgG2 backbones, and are therefore mixtures ofconformationally distinct isoforms resulting disulfide shuffling. Thehigh cell density mixed lymphocyte reaction (MLR) was chosen for thesestudies as previous data indicates that this assay provides a robustread out of functionality for both INX901 and INX908. The followingmodifications of INX901 and/or INX908 were made to investigate whetherspecific isoforms are responsible for function: biochemical skewing toeither the A or B isoform, genetic modifications to “lock” theconformation into the A or B form, and chimeric molecules where the Fcwas silenced and the CH1/hinge region came from either IgG1, in whichdisulfide shuffling does not occur, or IgG2, which allows for nativedisulfide shuffling.

The results of the assay indicate that INX901 and INX908 retain functionregardless of whether in the A form, B form, or the mixture of formsthat characterizes a native IgG2. Additionally, both INX901 and INX908require an active Fc region for functionality.

The MLR is a standard immunological assay that depends upon MHC class Iand II mismatching to drive an allogeneic T cell response. Peripheralblood mononuclear cells are isolated from two mismatched individuals,incubated together and as a result of these mismatches, proliferationand cytokine production occurs. High cell density conditions (HCD),meaning cultures with >1×10⁷ cells/ml, have previously been reported toelucidate agonistic functions of antibodies in vitro. Our previous dataindicates that both INX901 and INX908 can suppress the expression ofTNFα under HCD conditions in the MLR.

The HCD MLR assay was used to assess the function of INX901 and INX908following either genetic or biochemical modifications with respect toIgG2 disulfide isoforms and/or Fc silencing of each antibody. Prior torunning the MLR, each antibody was confirmed to bind recombinant VISTAvia ELISA. INX901 was sent to Elion, LLC (Louisville, Colo.) where itwas modified by redox to either be predominantly A form (INX901 A skew)or B form (INX901 B skew). Skewing was confirmed by RP-HPLC as describedin the prior example. (FIG. 11). Each antibody, as well as the parentalINX901, was diluted in a dose response in the HCD MLR and cytokineproduction was measured by Luminex. Previous data has indicated thatTNFα and/or IL-2 are robust readouts for antibody function of theparental INX901 antibody. In two separate MLRs, both TNFα and IL-2 werereduced by INX901 parental, INX901 A skew and INX901 B skew compared tothe IgG2 control (FIG. 13).

To confirm the data from FIG. 13, additional variants of INX901 weremade with mutations to generate locked variants in either the A form orthe B form. Additionally, chimeric versions of INX901 were made withfully silent Fc regions to test the function of the Fc domain. INX901 Siis a fully silent IgG1 antibody. INX901 HSi has a fully silent IgG1 Fc,but also possesses an IgG2 CH1/hinge region that enables disulfideshuffling that is indistinguishable from a native IgG2. Prior to runningthe MLR, each antibody was confirmed to bind recombinant VISTA viaELISA. Confirming the data from the biochemical skewing, both the A lockand B locked versions of INX901 were able to reduce the production ofboth IL-2 and TNFα (FIG. 14). In contrast, both the Si and HSi versionsof INX901 were unable to reduce production of IL-2 and TNFα (FIG. 14).

To confirm the data from FIG. 14, identical mutations were made to theINX908 antibody to generate locked variants in either the A form or theB form. Additionally, chimeric versions of INX908 were made with fullysilent Fc regions to test the function of the Fc domain. INX908 Si is afully silent IgG1 antibody. INX908 HSi has a fully silent IgG1 Fc butcontains the IgG2 CH1/hinge region. Prior to running the MLR, eachantibody was confirmed to bind recombinant VISTA via ELISA. Confirmingthe data with the INX901 variants, both the A lock and B locked versionsof INX908 was able to reduce the production of both IL-2 and TNFα (FIG.15). In contrast, both the Si and HSi versions of INX908 were unable toreduce production of IL-2 and TNFα (FIG. 15).

Example 15: Discontinuous Epitope Mapping of Agonist Antibodies UsingPEPPSCAN Methods

Pepscan uses peptide arrays to determine both linear and discontinuousepitopes. This methodology is an accepted method used by manyresearchers and companies to ascertain antibody epitopes. FIG. 16schematically describes the Pepscan® technology used to identify linearand discontinuous epitopes bound by various agonist anti-human VISTAantibodies according to the invention.

The Principles of Clips Technology

CLIPS technology structurally fixes peptides into definedthree-dimensional structures. This results in functional mimics of eventhe most complex binding sites. CLIPS technology is now routinely usedto shape peptide libraries into single, double or triple loopedstructures as well as sheet- and helix-like folds. The CLIPS reactiontakes place between bromo groups of the CLIPS scaffold and thiolsidechains of cysteines. The reaction is fast and specific under mildconditions. Using this elegant chemistry, native protein sequences aretransformed into CLIPS constructs with a range of structures.

Combinatorial Clips Library Screening in Detail

CLIPS library screening starts with the conversion of the target proteininto a library of up to 10,000 overlapping peptide constructs, using acombinatorial matrix design. On a solid carrier, a matrix of linearpeptides is synthesized, which are subsequently shaped into spatiallydefined CLIPS constructs. Constructs representing both parts of thediscontinuous epitope in the correct conformation bind the antibody withhigh affinity, which is detected and quantified. Constructs presentingthe incomplete epitope bind the antibody with lower affinity, whereasconstructs not containing the epitope do not bind at all. Affinityinformation is used in iterative screens to define the sequence andconformation of epitopes in detail. The results of this epitope analysisare summarized below.

Antibodies INX901, INX902, INX904, INX906, INX907, INX908

When tested under moderate stringency conditions antibodies INX901,INX902, INX904, INX906, INX907, INX908 strongly bound linear andconformational epitope mimics. Bound peptides contain core sequences₄₈NVTLTCRLLGPV₆₀ (SEQ ID NO:67), ₇₉EVQTCSERRPIR₉₀ (SEQ ID NO:68),₁₂₃SDHHGNFS₁₃₀ (SEQ ID NO:69) and ₁₅₃HHHSEH₁₅₈ (SEQ ID NO:70), wherepeptide stretch ₇₉EVQTCSERRPIR₉₀ (SEQ ID NO:68) is the dominant part ofthe epitope.

Additional analysis of data recorded with linear epitope mimics allowedus to identify residues that are important for binding for INX904,INX906, INX907 and INX908, as double Ala mutants on certain positionsnotably decreased signal intensities. In particular, replacement ofresidues CR within ₄₈NVTLTCRLLGP₆₀ (SEQ ID NO:71) affects binding ofINX906, INX907 and INX908. Also the replacement of residues TC within₇₉EVQTCSERRPIR₉₀ (SEQ ID NO:68) notably affects binding of INX904 andINX907.

Antibody INX800

When tested under moderate stringency conditions antibody INX800 did notdetectably bind linear and simple constrained epitope mimics, but showeddetectable binding with discontinuous epitope mimics. Analysis of dataobtained with discontinuous epitope mimics suggest that antibody INX800recognizes a discontinuous epitope with core sequences ₅₃TCRLLGPVDKG₆₈SEQ ID NO:72), 101HGGHQAA₁₀₇ (SEQ ID NO:73), ₁₂₁SASDHHGNFS₁₃₀ (SEQ IDNO:74) and ₁₅₃HHHSEHRVHGAM₁₆₄ (SEQ ID NO:75), where sequence₁₅₃HHHSEHRVHGAM₁₆₄ (SEQ ID NO:76) represents the dominant recognitionsite.

Antibodies INX803 and INX804

When tested under high stringency conditions antibodies INX803 andINX804 did not bind any peptide present on the array. When tested undermoderate stringency conditions both antibodies bound discontinuousepitope mimics. Cumulative analysis of binding profiles suggests thatboth antibodies similarly recognize peptide stretches ₅₂LTCRLLGPV₆₀ (SEQID NO:77), ₇₉EVQTCSERRPIR₉₀ (SEQ ID NO:78), ₉₈HLHHGGHQAA₁₀₇ (SEQ IDNO:79), ₁₂₃SDHHGNFS₁₃₀ (SEQ ID NO:80), ₁₅₃HHHSEHRVHGAM₁₆₄ (SEQ IDNO:81), where region ₅₂LTCRLLGPV₆₀ (SEQ ID NO:77) is the dominantrecognition site.

Antibody INX900

When tested under high stringency conditions antibody INX900 very weaklybound linear epitope mimics with core sequence ₇₉EVQTCSERRPIR₉₀ (SEQ IDNO:68). Notably higher binding was observed with discontinuous epitopemimics, which in addition to sequence ₇₉EVQTCSERRPIR₉₀ (SEQ ID NO:68)contain core sequences ₅₆LLGPVDKGHDVTFYK₇₀ (SEQ ID NO:82),₁₁₃LAQRHGLESASDHHG₁₂₇ (SEQ ID NO:83), ₁₅₃HHHSEHRVHGAM₁₆₄ (SEQ ID NO:84).

Antibody INX903

When tested under high stringency conditions antibody INX903 did notbind linear epitope mimics, but weakly bound conformational epitopemimics. Analysis of recorded intensity profiles suggests that theantibody recognizes a discontinuous epitope composed of core sequences₇₉EVQTCSERR₈₇ (SEQ ID NO:85), ₉₃TFQDLHLHHGGHQAA₁₀₇ (SEQ ID NO:86),₁₄₆CLVVEIRHHHSEH₁₅₈ (SEQ ID NO:87), where sequence ₇₉EVQTCSERR₈₇ (SEQ IDNO:85) is the core of the epitope.

Antibody INX905

When tested under high stringency conditions antibody INX905 boundlinear peptides with core sequence ₇₉EVQTCSERRP₈₈ (SEQ ID NO:88). Dataacquired with double Ala mutants indicate that motif RR within₇₉EVQTCSERRP₈₈ (SEQ ID NO:88) is critical for the recognition. Intensityprofiles recorded with discontinuous epitope mimics suggest thataddition of peptide sequences ₅₃TCRLLGPVDKG₆₃ (SEQ ID NO:89),₁₂₃SDHHG₁₂₇ (SEQ ID NO:90) and ₁₅₃HHHSEHRVHGAM₁₆₄ (SEQ ID NO:91)augments binding of the antibody. FIG. 17 shows that most agonistanti-human VISTA antibodies bind to the same core sequence. FIG. 18 alsosummarizes the epitope results. FIG. 19 shows the epitopes bound byagonist anti-human VISTA antibodies and identifies important residuesinvolved in binding.

Example 16: Use of Agonist Anti-VISTA Antibodies in TreatingInflammatory Bowel Disease

Intestinal inflammation is initiated with syngeneic splenic CD4⁺CD45^(R8h−) T cell adoptive transfer into T and B cell deficientrecipient mice. The CD4+ CD4

T cell population contains mainly naïve T cells primed for activationthat are capable of inducing chronic small bowel and colonicinflammation. This method provides precise initiation of disease onsetand a well-characterized experimental time course permitting the kineticstudy of clinical features of disease progression in mice. Intestinalinflammation induced by this method shares many features with human IBD,including chronic large and small bowel transmural inflammation,pathogenesis driven by cytokines such as TNF and IL-12, and systemicsymptoms such as wasting. Thus, it is an ideal model system for studyingthe pathogenesis of human IBD.

Materials & Methods

INX901 treatment was administered at days 0, 3, and 6 following DDE1CD4⁺; CD45RBhi spleen cell transfer. At day 14, peripheral blood wasanalyzed by flow cytometry. Mouse weight was recorded during the wholeexperiment. As soon as the mice started losing weight, at day 46 theexperiment was terminated and cardiac blood and spleen cells wereanalyzed by flow cytometry; colons were dissected and processed forhistology after being measured. There were two treatment groups: humanIgG2 and INX901 (8 mice each), as well as a control group (8 mice) thatreceived a whole CD4+ cell iv injection and is a negative control fordisease development.

Mice

Donor Mice:

Human VISTA knock-in (DDE1) mice have the human VISTA cDNA knocked-in inplace of the mouse VISTA gene, and express only human VISTA both at RNAand protein level. The mice are bred at Sage Labs (Boyertown, Pa.). Themice, aged 8-12 weeks, first transited for 3 weeks in the quarantinefacility, and then were transferred to our regular facility. 4-month oldmale DDE1 mice were used.

Recipient Mice:

9 weeks old male Rag1 Knockout mice were purchased from the Jackson Lab(B6.129S7-Rag^(1tm1Mom/j)).

Spleen Cell Isolation and Transfer

CD4⁺ T cells were isolated by negative selection using the StemCellEasySep™ Mouse Naïve CD4+ T cell isolation kit (#19765), skipping thememory T cell depletion step. One DDE1 spleen was mechanicallydissociated between 2 glass slides and CD4 T cells were isolatedfollowing manufacturer instructions. Briefly spleen cells wereresuspended in PBS 2% FBS, 1 mM EDTA. They were then incubated with ratserum and CD4 T cell isolation cocktail. After 7.5 min, magnetic beadswere added and incubated for 2.5 min. The tubes containing spleen cellsand beads are then placed onto a magnet and after 2.5 minutes, theunbound cells are transferred into a new tube. We obtained a total of11.3×10⁶ CD4 T cells. Each control mouse received intravenous (i.v.)injection of 0.5×10⁶ cells in 200 μl.

Naïve CD4 T Cell Transfer:

T cell isolation was conducted on 11 DDE1 spleens. Naïve CD4+ wereisolated by negative selection using the StemCell EasySep™ Mouse NaïveCD4+ T cell isolation kit (#19765) following manufacturer instructionsas described above adding the memory T cell depletion step.

To select CD45RB⁺ cells, CD4⁺ cells were resuspended at 100×10⁶ cells/mlin PBS 2% FBS, 2 mM EDTA (binding buffer) and anti CD45RB PE antibodyand incubated for 10 min at 4° C. After 2 washes in binding buffer, antiPE MicroBeads (Miltenyi #130-048-801) were added and incubated for 15min at 4° C. After 2 washes in binding buffer, cells were resuspended in1 ml of binding buffer and placed on 2 Miltenyi MS columns. Cells wereeluted in twice 1 ml per column. A total of 5.6×10⁶ CD4⁺ CD45RB⁺; cellswere obtained. Mice received by i.v. injection 0.329×10⁶ cells in 200μl.

Cell purity was evaluated by flow cytometry using the following panel:

-   -   CD4 APC-Cy7 (BioLegend at 1:200)    -   CD45RB PE (BioLegend at 1:200)    -   Yellow Live/Dead (Invitrogen at 1:1000)

Anti-Human VISTA Antibodies and Dosage

INX901 and control human IgG2 were dosed at 3 mg/kg. At day 0,antibodies were injected i.p. 3 hours before i.v. cell transfer intorecipient Rag1 KO mice. Mice were then dosed on day 3 and 6 viaintraperitoneal (i.p.) injections. Control group received human IgG2(Lot AB150073—4.7 mg/mL). Treated group received INX901 (LotBP-021-016-14—5.46 mg/mL).

Evaluation of “Absolute” Immune Cell Change in Peripheral Blood

At day 14 after donor cell transfer, donor CD4 T cell accumulation andactivation status were evaluated by flow cytometry on retro-orbitalbleed. Briefly, blood was collected in Eppendorf tubes containing 10 μlof heparin. To obtain a close to absolute cell count, 100 μl of wholeblood was stained by directly adding the antibody cocktail+mouse Fcblock in 10 μl. After 30 min at 4° C., 760 μl of ACK lysis buffer isadded to each sample. After 20 min incubation, samples are washed oncein PBS. Samples were run on a MACSQuant flow cytometer and analyzed withthe FlowJo program.

Antibody Cocktail

Fluorophore FITC PE PerCP Cy5.5 PE-Cy7 APC eFluor 780 BV421 BV510Antigen CD62L CD45RB CD11b CD25 PD1 CD44 CD4 CD45 Dilution 1:200 1:2001:200 1:200 1:200 1:200 1:200 1:200

Tissue Collection for Histology

On day 46, mice were euthanized and the colon dissected. After lengthmeasurement, the colons were emptied, transferred in cassettes. Aftero/n in 10% formalin, the cassettes were transferred into 70% ethanol andprocessed by the Pathology department for paraffin Embedded, Sectioningand H&E Staining.

Evaluation of Immune Cell Change in Spleen at Terminal Time Point

Briefly, spleens were collected and mechanically dissociated. FollowingACK lysis, cells were washed and stained with the following antibodycocktail. Samples were run on a MACSQuant flow cytometer and analyzedwith the FlowJo program.

Antibody Cocktail

PerCP Fluorophore FITC PE Cy5.5 PE-Cy7 APC eFluor780 BV421 BV510 AntigenCD62L CD45RB CD11b CD25 FoxP3 CD44 CD4 LD Yellow Dilution 1:200 1:2001:200 1:200 1:200 1:200 1:200 1:1000

INX901 Treatment Leads to Decreases in CD4 T Cell Numbers in PeripheralBlood

Analysis of immune cell changes in peripheral blood at day 14 showeddecreases in CD4 T cells both as absolute count or as a fraction of CD5⁺cells and the CD4 T cells that remained were mainly CD45RB^(hi) (FIG.20). As shown therein absolute numbers in 100 μl of blood (left graph);frequencies of CD45⁺ cells (center graph); frequencies of CD4+ cells(right graph) (n=8 per group, SEM, statistic unpaired T-test, no equalSD). The small population CD4 T cells that were present at day 14 postINX901 treatment appeared to be still naïve as shown by thestatistically higher expression level of CD62L and CD45RB (FIG. 21). Thefigure shows changes in CD4 T cell activation status in peripheralblood. (n=8 per group, SEM, statistic unpaired T-test, no equal SD)(MFI: median fluorescence intensity).

By day 30, the mice that received CD45R

naïve CD4 T cells started losing weight as colitis progressed while thecontrol group that received whole T cell fraction did not develop anydisease (FIG. 22). INX901 treatment prevented the weight loss and miceappeared to gain weight instead when compared to control group. Micewere euthanized at day 46 and colon and spleen collected for histologyand flow cytometry respectively.

Further, mice suffering from colitis showed a shortening of the colonwhen compared to the control (total CD4) group, no shortening occurredin the group treated with INX901. As shown in FIG. 23, while micesuffering from colitis showed a shortening of the colon when compared tothe control (total CD4) group, no shortening occurred in the grouptreated with INX901. As additionally shown in FIG. 24, mice that weresubjected to CD4 CD45R

cell transfer all developed colitis as shown by the presence ofimportant inflammatory infiltrates between intestinal villi (arrows,middle pictures at low and high magnification) when compared to thecontrol group that received total cell transfer and subsequently did notdevelop any colitis (upper pictures). INX901 treatment completelyprevented the development of colitis as shown by the complete absence ofinflammatory infiltrate in all the samples analyzed (8 mice per group).

As shown in the figure INX901 treatment prevented colitis development.Representative pictures of H&E stained sections of the colon for eachmouse group. Magnification: pictures on the top are at 4×, on the bottomat 20×. Arrows indicate areas with abundant inflammatory infiltrates.Note their complete absence in the INX901-treated colon sample.

Immunohistochemistry staining of CD3 (FIG. 25) and CD11b (FIG. 26)expressing cells confirmed colitis development in the IgG2 treatedanimals. Similar numbers of CD3+ and CD11b+ cells in control andINX901-treated samples again show the complete absence of diseasefollowing INX901 treatment. FIG. 25 shows that INX901 treatmentprevented CD3+ T cell recruitment to the colon. Representative picturesof CD3 stained sections of the colon for each mouse group.(Magnification: pictures on the top are at 4×, on the bottom at 20×).

FIG. 26 shows that INX901 treatment prevented myeloid (CD11b+) cellrecruitment to the colon. Representative pictures of CD11b stainedsections of the colon for each mouse group. Magnification: pictures onthe top are at 4×, on the bottom at 20×. Yet additionally, INX901treatment further was shown to induce long term CD4 T cell changes.Analysis by flow cytometry on spleen cells showed that 40 days postINX901 treatment, there was still highly significant decreases in CD4 Tcell frequencies as compared to the IgG2 treated group, and 10 to 30% ofthe CD4 T cells were still CD45RB+ (FIG. 27), left and right graphs)).No changes in regulatory T cells were observed following INX901treatment (FIG. 27, center graph). As shown in the figure spleens werecollected at day 46 (40 days post last antibody dosage) and analyzed byflow cytometry (n=8 or 4 per group, SEM, statistic unpaired T-test, noequal SD).

Accordingly we have shown that anti-VISTA INX901 treatment preventscolitis development as shown by the absence of weight loss, change incolon length and inflammatory infiltrate present in the colonAdditionally, we observed long-term decreases in CD4 T cells (at 14 and40 days post treatment) that retained naïve T cell characteristics suchas CD45RB expression. Therefore, anti-VISTA agonist antibodies may beused to treat or prevent colitis.

Example 17: Use of Agonist Anti-VISTA Antibodies in Treating PsoriasisImiquimod (IMQD) Induced Psoriasis Model

Imiquimod (IMQD) is a commercially available cream containing TLR7/8agonists that is widely used for dermatological conditions such as viralinfections and melanoma. Application of IMQD to the skin over multipledays results in thickening of the epidermis via proliferation of thekeratinocytes. Additionally, an immunological infiltration into thedermis layer occurs, with populations of both T cells and myeloid cells.Recurrent administration of IMQD creates a skin lesion similar to whatis observed in patients with Psoriasis. IL-17 and IL-23 are thought tobe the major cytokines involved in the immune response to IMQD.

In these experiments, we test the function of 8G8, a hamster α mouseVISTA antibody, on the IMQD induced Psoriasis. Mice were dosed everyother day with 8G8, while IMQD was administered topically to the back ofmice for 7 days. The skin was then isolated, fixed and embedded inparaffin blocks. Sections were then stained for H&E analysis andexpression of several immunological populations by IHC. Notably, 8G8drastically reduced the overall cellular infiltrate into the dermis,much of which appears to be a reduction in the CD3+ population.

Materials and Methods Mouse Treatment

1. 7-week old mice Balb/c mice were purchased from Jackson and stored inSPF conditions at the DHMC. The backs of the mice were shaved prior tothe start of the experiment.2. Imiquimod was purchased from the Dartmouth Hitchcock animal facility.62.5 mg was applied to skin daily through the use of a Q-tip.3. 8G8 was administered every other day at 200 μg/mouse.4. Mice were sacrificed at day 7 and the skin was isolated by cutting asquare from the shaved section. The skin was placed in Formalin for 24hours before being delivered to the Dartmouth Pathology department forembedding in Paraffin.

Splenic Analysis

1. The spleen was isolated and ground into a single cell suspension.2. After centrifugation, the red blood cells were lysed using ACKsolution (5 minutes at RT).3. Cells were centrifuged and resuspended in PBS before counting.4. 1×10⁵ cells from each spleen were labeled with CD4, CD8, CD19, CD11b,Ly6C and Ly6G in the presence of mouse Fc block, at 20 minutes on ice.5. Labeled cells were run on the Miltenyi MACSquant and analyzed usingFlowJo.6. Statistics were performed in PRISM 6, with groups compared by one-wayANOVA followed by Sidak's multiple comparisons test. P-values aredenoted as follows: p<0.0001****, p<0.001***, p<0.01**, p<0.05*.

H&E and IHC Analysis

1. Samples were put into cassettes and fixed O/N in 10% Formalin at roomtemperature, then briefly washed in PBS and transferred and kept into70% Ethanol (Fisher Scientific) prior to being transferred to thePathology Translational Research Core at the Geisel School of Medicineat Dartmouth where they were paraffin embedded, sectioned and thenstained.2. Paraffin embedded tissue sections (4 μm) were stained using a LeicaBOND RX automated stainer. After dewaxing, the sections were subjectedto antigen retrieval (Bond epitope retrieval solution 2, 100° C., 20min) and incubated with the primary antibody (see dilution below) for30-60 min, at room temperature in Leica diluent. Slides are then washed3×5 min washes in PBS and incubated with secondary antibody (from LeicaBond Refine detection kit, DS9800). After 3 final washes in PBS thesections were incubated with DAB (Leica Bond polymer detection kit),rinsed, counterstained with hematoxylin and mounted.3. Statistics on CD3+ infiltration were performed in R statisticalprogramming language. The chi-squared test for outlier (in the“outliers” package) was performed for each group, and if p<0.05 thatdata point was removed. Groups were then compared using Student's t-testwith pooled variance, and p<0.05 denoted as *.

Speci- Ig Clone/ Catalog Re- Di- ficity type Format # Company trievallution mouse rabbit Polyclonal AB5690 Abcam EDTA 0.25 CD3 mouse rabbitclone 1 50134- SinoBiologicals EDTA 0.32 CD4 R001 mouse rabbitPolyclonal A0398 Dako EDTA 0.74 MPO mouse rabbit Polyclonal ab- AbcamEDTA 0.25 CD11b 75476 mouse rabbit clone NBP2- Novus EDTA 0.11 F4/80SP115 12506

Results

We determined whether an agonist anti-VISTA antibody would be effectivein a psoriasis model, particularly we determined if an agonistanti-human antibody, 8G8, was capable of altering the immunologicalresponse to Imiquimod induced Psoriasis. IMQD cream was administered tothe back skin of Balb/c mice every day for 7 days. Mice were given 200μg of 8G8 or Hamster Ig at the time of initial IMQD treatment (day 0),and at day 2, day 4 and day 6. The mice were sacrificed at day 7 and theskin was analyzed by H&E and IHC for several immune subset markers.

The H&E analysis indicated a reduced number of dense nucleated cells(lymphocyte characteristic) into the dermis of 8G8 treated mice comparedto controls (see FIG. 28). We therefore performed IHC analysis forseveral cell surface markers expressed by immunological cells. Of note,the number of CD3⁺ cells was reduced in the 8G8 treated mice compared tothe Hamster Ig control group (see FIG. 29, 30). IMQD Psoriasis isthought to be canonically driven by T cells, specifically IL-17producing populations including Th17 and γδ T cells.

We also profiled the splenic populations to see whether 8G8 treatmentreduced global T cell populations, or if they CD3⁺ decreases were justobserved in the skin. No changes to the T cell populations wereobserved, however small decreases in CD11b⁺ Ly6G (% of total) and CD19+(total number) populations did occur with 8G8 (see FIG. 31).

Therefore, these experiments revealed that an anti-human VISTA agonistantibody was effective in a psoriasis model as 8G8 was shown to reducethe number of CD3⁺ T cells infiltrating Imiquimod treated skin. Based onthese results VISTA agonist antibodies may be used in the treatment orprevention of psoriasis and other T cell mediated autoimmune orinflammatory skin conditions.

Example 18: Use of Agonist INX800 and INX801 Anti-VISTA Antibodies inConcanavalin A Induced Hepatitis Model

Concanavalin A (ConA) is a lectin that binds to specific sugarsultimately leading to the activation of the immune system, mainly in theliver. ConA induces rapid production of multiple cytokines, such as Tcell derived IL-2, IL-3, 11-4, TNF-α and IFN-γ. The T cell activationand subsequent cytokine response induces acute hepatitis, and in highdose models, mortality. ImmuNext has created a human VISTA knock-inmouse where human VISTA is expressed in replacement of mouse VISTA. Themice are phenotypically normal, indicating that hVISTA functionsappropriately in the mice. We have therefore tested two anti-human VISTA

Antibodies, Called INX800 and INX801 in the ConA Model of Hepatitis inthese Mice. MATERIALS AND METHODS

10-week old mice were purchased from Jackson and stored in SPFconditions at the DHMC.

Concanavalin A from Canavalia ensiformis (Sigma, cat # C2010-100MG) wasreconstituted in 10 ml of fresh PBS and shaken for 30 minutes at RT. 300μL aliquots were made for storage at −20° C.

200 μL of α-hVISTA antibodies, or the Control Ig control (CrownBiosciences) were administered to mice 3 hours prior to ConA treatmentby I.P injection in PBS. Antibodies were given at 10 mpk.

Animals were weighed and given either 15 mg/kg of ConA for the cytokineanalysis experiments. Injections were performed through the tail vein.In all cases, mice were monitored every several hours for morbidity andsacrificed if cold to touch.

For the cytokine analysis, mice were bled by sacrifice using C02 andthen cardiac puncture. Blood was collected in plasma collection tubesand then centrifuged to remove the cellular component. Samples werestored for short periods of time at −80° C. before being analyzed bymultiplex per manufacturer's instructions.

Calibration curves from recombinant cytokine standards were preparedwith threefold dilution steps in the same matrix as the samples.

High and low spikes (supernatants from stimulated human PBMCs anddendritic cells) were included to determine cytokine recovery.

Standards and quality controls were measured in technical triplicate,each triplicate test sample was measured once, and blank values weresubtracted from all readings. All assays were carried out directly in a96-well filtration plate (Millipore, Billerica, Mass.) at roomtemperature and protected from light.

Briefly, wells were pre-wet with 100 μl PBS containing 1% BSA, thenbeads together with a standard, sample, spikes, or blank was added in afinal volume of 100 μl, and incubated together at room temperature for30 min with continuous shaking.

Beads were washed three times with 100 μl PBS containing 1% BSA and0.05% Tween 20.

A cocktail of biotinylated antibodies (50 μl/well) was added to beadsfor a 30-min incubation at room temperature with continuous shaking.

Beads were washed three times, then streptavidin-PE was added for 10min. Beads were again washed three times and resuspended in 125 μl ofPBS containing 1% BSA and 0.05% Tween 20.

The fluorescence intensity of the beads was measured using the Bio-Plexarray reader. Bio-Plex Manager software with five parametric-curvefitting was used for data analysis.

Statistics for the cytokine analysis were carried out in R StatisticalComputing Language or in Prism 6. Cytokine concentration values belowdetection (<OOR) were rescaled to the lowest detectable concentration,and values above accurate qua ntitation (>OOR) were rescaled to themaximum linearly quantifiable concentration. Pair-wise comparisonsbetween the antibody-treated groups and an Ig-control were made usingOne-Way ANOVA with Tukey Honest Significant Differences. P-values lessthan 0.1 were deemed significant and denoted as follows: p<0.0001****,p<0.001***, p<0.01**, p<0.05*, p<0.1˜.

Results

Two replicate experiments (#13 and #14) were designed to determinewhether INX800 and/or INX801, both anti-hVISTA antibodies, were capableof altering the cytokine response to ConA. Mice were given 10 mpk ofINX800, INX801 or Control Ig three hours prior to treatment with ConA(15 mg/kg) and then mice were sacrificed at the 6-hour time point andthe cytokine response was determined by 32-plex. Most cytokines wereunchanged, however IL-2 was consistently decreased by both INX800 andINX801 (FIGS. 32, 33). All cytokine profiles can be seen in the linksfound in the Appendix.

The naïve mice and mice from experiment 14 were also analyzed forchanges in immune populations to determine whether the decrease incytokines could be associated with any sign of T cell depletion. Theflow gating strategy included markers for B220 (B cells); CD3, CD4, andCD8 (T cells); and CD11b and Gr1 (myeloid cells). No statisticallysignificant association in the total number of cells with antibodytreatment was observed for any population (See FIG. 34). Therefore,INX801 and INX801 both are capable of suppressing a ConA induced IL-2response. There is no obvious sign of cellular depletion caused byINX800 or INX801 either in naïve mice or during the ConA response. Theseexperimental results suggest that VISTA agonist antibodies may be usedto treat and prevent hepatitis infection and inflammation and cytokineresponses elicited during acute infection. These experimental resultssuggest that VISTA agonist antibodies may be used to treat and preventhepatotoxicity or liver damage associated with hepatitis and otherinfections and inflammatory diseases that affect the liver.

Example 19: Use of Agonist Anti-VISTA 8G8 and 13F3 Antibodies inConcanavalin a Induced Hepatitis Model

The ConA model is described in the prior example. In these experimentswe examined the ability of two different α-mVISTA antibodies (8G8 and13F3) on the cytokine responses induced by ConA. As 8G8 was able todecrease production of several T cell derived cytokines, we alsoexamined whether it could protect against a lethal dose of ConA.

Materials & Methods

7-week old mice were purchased from Jackson and stored in SPF conditionsat the DHMC.

Concanavalin A from Canavalia ensiformis (Sigma, cat # C2010-100MG) wasreconstituted in 10 ml of fresh PBS and shaken for 30 minutes at RT. 300μL aliquots were made for storage at −20° C.

200 μL of α-mVISTA antibodies 8G8 and 13F3, or the Hamster Ig control(BioXcell) were administered to mice 3 hours prior to ConA treatment byI.P injection in PBS.

Animals were weighed and given either 15 mg/kg of ConA for the cytokineanalysis experiments or 30 mg/kg of ConA for the mortality experiments.Injections were performed through the tail vein. In all cases, mice weremonitored every several hours for morbidity and sacrificed if cold totouch.

For the cytokine analysis, mice were bled either by cheek puncture or bysacrifice using C02 and then cardiac puncture. Blood was collected inplasma collection tubes, incubated for 30 min at RT and then centrifugedto remove the cellular component. Samples were stored for short periodsof time at −80° C. before being analyzed by multiplex per manufacturer'sinstructions.

Calibration curves from recombinant cytokine standards were preparedwith threefold dilution steps in the same matrix as the samples.

High and low spikes (supernatants from stimulated human PBMCs anddendritic cells) were included to determine cytokine recovery.

Standards and quality controls were measured in technical triplicate,each triplicate test sample was measured once, and blank values weresubtracted from all readings. All assays were carried out directly in a96-well filtration plate (Millipore, Billerica, Mass.) at roomtemperature and protected from light.

Briefly, wells were pre-wet with 100 μl PBS containing 1% BSA, thenbeads together with a standard, sample, spikes, or blank were added in afinal volume of 100 μl, and incubated together at room temperature for30 min with continuous shaking.

Beads were washed three times with 100 μl PBS containing 1% BSA and0.05% Tween 20.

A cocktail of biotinylated antibodies (50 μl/well) was added to beadsfor a 30-min incubation at room temperature with continuous shaking.

Beads were washed three times, then streptavidin-PE was added for 10min. Beads were again washed three times and resuspended in 125 μl ofPBS containing 1% BSA and 0.05% Tween 20.

The fluorescence intensity of the beads was measured using the Bio-Plexarray reader. Bio-Plex Manager software with five parametric-curvefitting was used for data analysis.

Statistics for the cytokine analysis were carried out in R StatisticalComputing Language or in Prism 6. Cytokine concentration values belowdetection (<OOR) were rescaled to the lowest detectable concentration,and values above accurate quantitation (>OOR) were rescaled to themaximum linearly quantifiable concentration. Pair-wise comparisonsbetween antibody-treated groups and the Ig-control were made usingOne-Way ANOVA with Tukey Honest Significant Differences. P-values lessthan 0.05 for all tests and comparisons were deemed significant.

Results

Our initial experiment was designed to determine whether 8G8 or 13F3,both anti-mVISTA antibodies, were capable of altering the cytokineresponse to ConA. Mice were given 200 μg of 8G8, 13F3 or Hamster Igthree hours prior to treatment with ConA (15 mg/kg) and then mice weresacrificed at the 6-hour time point and the cytokine response wasdetermined by 32-plex (FIG. 35). Most cytokines were unchanged, howeverIL-2 was decreased in the presence of 8G8 but not 13F3. We then wantedto determine if the reductions in IL-2 could be correlated withprotection for ConA-induced mortality. To do so, mice were pre-treatedwith 8G8 or Hamster-Ig and then given ConA at 30 mg/kg and followed forsurvival (FIG. 36). While all of the Hamster Ig treated mice had to beeuthanized within 40 hours, 80% of the 8GB8 treated mice survived past72 hours and appeared healthy. Therefore, 8G8, the agonist antibody butnot 13F3, can induce changes to IL-2 in the ConA induced hepatitismodel. Further 8G8 protects against a lethal challenge of ConA (30mg/kg). These experimental results suggest that VISTA agonist antibodiesmay be used to treat and prevent hepatitis infection and pathologicalinflammation and proinflammatory cytokine responses elicited duringacute or chronic infection. These experimental results further suggestthat VISTA agonist antibodies may be used to treat and preventhepatotoxicity or liver damage associated with hepatitis and otherinfections and inflammatory diseases that affect the liver.

Example 20: Use of Agonist Anti-VISTA INX903 Antibody In Concanavalin AInduced

Hepatitis Model

The ConA model is described in the prior example. In these experimentswe have tested an additional anti-human VISTA antibody, INX903 in theConA model of hepatitis, using INX800 as a control. In one experiment,we also compared INX903 and INX800 to an antagonistic VISTA antibody,which has previously been shown to enhance cytokine production by immunecells.

Materials And Methods

10-week old mice were purchased from Jackson and stored in SPFconditions at the DHMC.

Concanavalin A from Canavalia ensiformis (Sigma, cat # C2010-100MG) wasreconstituted in 10 ml of fresh PBS and shaken for 30 minutes at RT. 300μL aliquots were made for storage at −20° C.

200 μL of α-hVISTA antibodies, or the Control Ig control (CrownBiosciences) were administered to mice 3 hours prior to ConA treatmentby I.P injection in PBS. Antibodies were given at 10 mpk.

Animals were weighed and given either 15 mg/kg of ConA for the cytokineanalysis experiments. Injections were performed through the tail vein.In all cases, mice were monitored every several hours for morbidity andsacrificed if cold to touch.

For the cytokine analysis, mice were bled by sacrifice using C02 andthen cardiac puncture. Blood was collected in plasma collection tubesand then centrifuged to remove the cellular component. Samples werestored for short periods of time at −80° C. before being analyzed byLuminex or MSD multiplex per manufacturer's instructions.

Statistics for the cytokine analysis were carried out in R StatisticalComputing Language. Cytokine concentration values below detection (<OOR)were rescaled to the lowest detectable concentration, and values aboveaccurate quantitation (>OOR) were rescaled to the maximum linearlyquantifiable concentration. Pair-wise comparisons betweenantibody-treated groups and the Ig-control were made using One-Way ANOVAwith Tukey Honest Significant Differences. P-values less than 0.1 weredeemed significant and denoted as follows: p<0.0001****, p<0.001***,p<0.01**, p<0.05*, p<0.1˜.

The results of these experiments are contained in FIG. 37 and FIG. 38.In FIG. 37 IL-2 expression was detected in the plasma from the 6-hourtime point of mice treated with Control-Ig, INX800 or INX903. Mice werepretreated at −3 hours with each of the indicated antibodies. At time 0,mice were dosed with 15 mg/kg of ConA, and then sacrificed and bled atthe 6-hour time point. FIG. 38 shows IL-2 and MIP-1 (3 expression in theplasma from the 6-hour time point of mice treated with Control-Ig, 2agonist VISTA abs (INX800 and INX903) or a VISTA antagonist Ab. Micewere pretreated at −3 hours with each of the indicated antibodies. Attime 0, mice were dosed with 15 mg/kg of ConA, and then sacrificed andbled at the 6-hour time point. These experiments show that INX800 andINX903 are capable of suppressing ConA induced cytokine responses.

These experimental results suggest that VISTA agonist antibodies may beused to treat and prevent hepatitis infection and pathologicalinflammation and proinflammatory cytokine responses elicited duringacute or chronic infection. These experimental results further suggestthat VISTA agonist antibodies may be used to treat and preventhepatotoxicity or liver damage associated with hepatitis and other acuteor chronic infections and inflammatory diseases that affect the liver.

Example 21: Use of Agonist 868 Anti-VISTA Antibody in Collagen InducedArthritis or CIA Model

Immunization of rodents and primates with Collagen type II (CII) inadjuvant induces an autoimmune arthritis, the so-called Collagen inducedarthritis or CIA that, in many ways, reproduces Rheumatoid Arthritis(RA) symptoms. CII is the major constituent protein of the cartilage ofdiarthrodial joints, the site of inflammation in RA, and immunity to CIIcan be detected in RA patients.

A cocktail of 5 monoclonal antibodies (Arthrogen-CIA® Arthritogenic5-Monoclonal Antibody Cocktail) recognizing the conserved epitopes onvarious species of CII can induce arthritis in naïve mice. This model iscalled Collagen Antibody-induced Arthritis (CAIA). In vitro studies withthe 5-Antibody cocktail showed that these antibodies could be pathogenicto chondrocytes even in the absence of inflammatory mediators, andimpair cartilage formation; they also inhibit collagen synthesis,fibrillogenesis and cause disorganization of CII fibrils in theextracellular matrix with or without increased matrix synthesis.Furthermore, the 5-antibody cocktail also has deleterious effects on thepre-formed cartilage.

In the present experiments, we tested the impact of 8G8 (hamsteranti-mouse VISTA monoclonal antibody) treatment on CAIA mouse model ofRA. Mice were dosed with 8G8 every other day starting at day −2. Theywere administered the 5-antibody cocktail on day 0 and LPS on day 3.Disease development was assessed by measuring inflammation swelling inthe affected joints over time. Clinical scoring was accomplished byawarding a score of 1 for each swollen digit, a score of 5 for a swollenfootpad and a score of 5 for a swollen wrist or ankle (Charles RiverLabs scoring system), which added together give a maximal score of 60for each animal.

Materials and Methods

Mouse Treatment

The DBA mice, 8-week old, were obtained from Jackson Labs. They wereacclimated for 2 days prior to having their tails tattooed.

CAIA induction: On day 0, mice were injected intraperitoneally (ip) withthe 5-antibody cocktail purchased from AMSBIO/Chondrex at a dosage of1.5 mg per mouse. Then on day 3, they received ip 50 μg of LPS (fromAMSBIO).

Anti-VISTA treatment: Mice were dosed every other day, starting at day−2, with anti-VISTA 8G8 or control hamster IgG at a dosage of 10 mg/Kgduring the whole course of the experiment. (see FIG. 39)

Statistical Analysis

CAIA scoring was analyzed using Excel for data management and GraphPadPrism for graphing. Statistical analysis was performed using a macro forR statistical computing software that measures divergence in tumorvolume between two groups of differentially treated mice and is named‘mixed effect repeated measures’.

Results

The objective of the experiment was to determine if an agonistanti-VISTA antibody would be effective in an arthritis model. In theexperiments it was shown that the anti-VISTA agonist antibody 8G8 couldaffect disease progression in the CAIA experimental model of RA. Thedata in FIG. 40 show significant decreases in disease progression andscope in response to 8G8 (interaction term P<0.000005). As showntreatment was initiated at day −2 and subsequently mice were dosed everyother day. (n=10 in each group). 8G8 treatment significantly reduceddisease severity (interaction term P<0.000005).

Example 22: Use of Agonist (INX903) Anti-VISTA Antibody in CollagenInduced Arthritis or CIA Model

The CIA model is described in the prior example. In the presentexperiment, we tested the impact of INX903 (human anti-human VISTA-IgG2)treatment on CAIA mouse model of RA. Mice were dosed with INX903 everyother day starting at day −2. They were administered the 5-antibodycocktail on day 0 and LPS on day 3. Disease development was assessed bymeasuring inflammation swelling in the affected joints over time.Clinical scoring was accomplished by awarding a score of 1 for eachswollen digit, a score of 5 for a swollen footpad and a score of 5 for aswollen wrist or ankle (Charles River Labs scoring system), which addedtogether give a maximal score of 60 for each animal.

Materials and Methods

Mouse Treatment

The experimental protocol is shown schematically in FIG. 41.

The hVISTA knock-in (KI) mice are bred at Horizon Discovery (Sage) Labs(Boyertown, Pa.). The mice, aged 8-12 weeks, first transit for 3 weeksin the quarantine facility, and then are transferred to the regularfacility. 4 month-old mice were used for this experiment. Mice got theirtail tattooed 2 days before experiment start.

CAIA induction: On day 0, mice were injected intraperitoneally (ip) withthe 5-antibody cocktail purchased from AMSBIO/Chondrex at a dosage of 5mg per mouse. Then on day 3, they received ip 50 μg of LPS (fromAMSBIO).

Anti-VISTA treatment: Mice were dosed every other day, starting at day−2, with anti-VISTA INX903 or control human IgG2 at a dosage of 10 mg/Kgduring the whole course of the experiment.

As shown in FIG. 42 treatment was initiated at day −2 and subsequentlymice were dosed every other day. (n=9 in control group and 8 in INX903treated group; 1 mouse was removed from the control group as it nevershowed any signs of disease). INX903 treatment significantly reduceddisease severity (interaction term P=0.0005). CAIA scoring was analyzedas in the prior example. The data shown in FIG. 42 indicate that thereis significant decreases in disease progression and scope in response toINX903 (interaction term P=0.0005).

Example 23: Use of Agonist (INX903) Anti-VISTA Antibody in CollagenInduced Arthritis or CIA Model

In the present experiment, we again tested the impact of INX903 (humananti-human VISTA-IgG2) treatment on CAIA mouse model of RA. Mice weredosed with INX903 every other day starting at day −2. They wereadministered the 5-antibody cocktail on day 0 and LPS on day 3. Diseasedevelopment was assessed by measuring inflammation swelling in theaffected joints over time. Clinical scoring was accomplished by awardinga score of 1 for each swollen digit, a score of 5 for a swollen footpadand a score of 5 for a swollen wrist or ankle (Charles River Labsscoring system), which added together give a maximal score of 60 foreach animal.

Materials and Methods

Mouse Treatment

The hVISTA knock-in (KI) mice are bred at Horizon Discovery (Sage) Labs(Boyertown, Pa.). The mice, aged 8-12 weeks, first transit for 3 weeksin the quarantine facility, and then are transferred to the regularfacility. 4 month-old mice were used for this experiment. Mice got theirtail tattooed 2 days before experiment start.

CAIA induction: On day 0, mice were injected intraperitoneally (ip) withthe 5-antibody cocktail purchased from AMSBIO/Chondrex at a dosage of 5mg per mouse. Then on day 3, they received ip 50 μg of LPS (fromAMSBIO).

Anti-VISTA treatment: Mice were dosed every other day, starting at day−2, with anti-VISTA INX903 or control human IgG2 at a dosage of 10 mg/Kgduring the whole course of the experiment as shown below.

CAIA scoring was analyzed as in the prior example.

The data shown in FIG. 43 show there to be significant decreases indisease progression and scope in response to INX903 (interaction termP=0.01).

Example 24: Use of Agonist (8G8) Anti-VISTA Antibody in Collagen InducedArthritis or CIA Model

In the present experiments, we tested the impact of 8G8 (hamsteranti-mouse VISTA monoclonal antibody) treatment on CAIA mouse model ofRA. Mice were dosed with 8G8 (agonist anti-murine VISTA antibody) everyother day starting at day −2. They were administered the 5-antibodycocktail on day 0 and LPS on day 3. Disease development was assessed bymeasuring inflammation swelling in the affected joints over time.Clinical scoring was accomplished by awarding a score of 1 for eachswollen digit, a score of 5 for a swollen footpad and a score of 5 for aswollen wrist or ankle (Charles River Labs scoring system), which addedtogether give a maximal score of 60 for each animal. As shown in FIG. 44the 8G8 antibody resulted in significant decreases in diseaseprogression and scope in response to 8G8 (interaction term P<0.0001.

Example 25: Use of Agonist Antibodies (INX800, 901 and 902) on DiseaseProgression in the Collagen Antibody Induced Rheumatoid Arthritis MouseModel

We tested the impact of INX800 (chimeric mouse anti-human VISTA-IgG2),INX901, and INX902 (human anti human VISTA-IgG2) treatment on CAIA mousemodel of RA. Mice were dosed with INX800, INX901, or INX902 every otherday starting at day −2. They were administered the 5-antibody cocktailon day 0 and LPS on day 3. Disease development was assessed by measuringinflammation swelling in the affected joints over time. Clinical scoringwas accomplished by awarding a score of 1 for each swollen digit, ascore of 5 for a swollen footpad and a score of 5 for a swollen wrist orankle (Charles River Labs scoring system), which added together give amaximal score of 60 for each animal.

CAIA scoring was analyzed as previously described. As shown in FIG. 45INX800 treatment qualitatively decreases disease progression, althoughnot with statistical significance in this experiment (interactionP=0.46) By contrast, as shown in FIG. 46, INX901 treatment fullyprevented disease progression (interaction P<0.01). As further shown inFIG. 47, INX902 treatment fully prevented disease progression(interaction P<0.0001).

Example 26: Use of Agonist VISTA Antibody (8G8) in C57/BI6 GVHD Model

The most commonly studied mouse model of MHC-mismatched acute GvHD isC57/BI6 (H2b)→BALB/c (H2d) (transplantation of cellular isolates fromC57/BI6 (H2b) donors into BALB/c (H2d) recipients). Here we used C57/BI6mice as donor for spleen cells and bone marrow transferred in irradiatedBALB/c recipient. We examined the immune-suppressive efficacy of Hamsterα-mouse VISTA antibody 8G8 compared to Hamster α-mouse VISTA antibody13F3 and Hamster IgG control.

Materials and Methods

10-week old females BALB/c recipient mice and C57/BI6 donor mice werepurchased from Charles River Laboratories. All mice were housed in SPFconditions at the DHMC vivarium.

Recipient mice were subjected to total body irradiation (TBI) emanatingfrom a cesium-137 source twice at 450 cGy at D0 (9.30 am and 1.30 μm)prior transfer

Donor mice were euthanized and bone marrow was harvested by flushingfemur and tibia with HBSS. Red blood cells were lysed using ACTsolution. Single cell suspension prepared isolated from spleens and redblood cells were lysed using ACT solution.

Recipient mice received 10 million bone marrow cells and 10 millionspleen cells along with 200 ug Hamster IgG (BioXcell, BE0091 lot#18206/1015) or 8G8 (lot# AB-130318) or 13F3-2E9 (lot# BP-075-014).Cells and antibodies were administered by tail vein intravenousinjection.

Three additional doses of antibodies were injected IP at D2, 4 and 6.

Mice were weighed regularly to monitor disease progression. However, allmice lost a lot of weight due to irradiation sickness and were providedliquid recovery food for the duration of the experiment. Mice wereeuthanized when showing signs of morbidity.

Results

FIG. 48A-B shows weights and survival of recipient mice treated with8G8, 13F3, or control Hamster IgG antibodies in acute GvHD diseasemodel. FIG. 48A shows mice appearance at day 21 and FIG. 48B showssurvival. As shown the hamster α-mouse VISTA antibody 8G8 isimmune-suppressive and strongly attenuates disease severity asillustrated by mice fur appearance at day 21 (FIG. 48a ) and protectsagainst GvHD induced lethality (FIG. 48b ). In contrast, 13F3 did notshow any such protection. Thus hamster 8G8 α-mouse VISTA isimmune-suppressive and strongly attenuates acute GvHD severity promotinglong-term survival. In contrast, 13F3 did not alter disease progression.

Example 27: Use of Agonist VISTA Antibodies (INX901, INX902, INX903 andINX904) in C57/BI6 GVHD Disease Model

We examined the immune-suppressive efficacy of α-human VISTA antibodies,INX901, INX902, INX903 and INX904, compared to Human Ig control bymeasuring their ability to modulate disease progression/severity in aC57/BI6 GvHD model. We also verified the presence of donor T-cells andcomplete chimerism in the surviving mice by flow cytometry of peripheralblood taken from the recipient mice at 41 days post-treatment.

Materials & Methods

9-11-week old males BALB/c recipient mice were purchased from Jackson.11-weeks old males Human-KI VISTA donor mice (DDE1) on C57/BI16background were purchased from Sage labs. All mice were housed in SPFconditions at the DHMC vivarium.

Recipient mice were subjected to total body irradiation (TBI) emanatingfrom a cesium-137 source at 300 cGy at Day−1 and D0 prior transfer.

Donor mice were euthanized and bone marrow was harvested by flushingfemur and tibia with HBSS. Red blood cells were lysed using ACKsolution. T cells were isolated from spleens by negative selection(Stemcell #19851).

Recipient mice received 10 million bone marrow cells and 2 million Tcells along with 10 mg/kg human IgG2 (Crown Bioscience, lot #AB150073)or INX901 (lot# BP-021-016-2), INX902 (lot# BP-021-016-3), INX903 (lot#BP-021-016-4) or INX904 (lot# BP-021-016-5). Cells and antibodies wereadministered by tail vein intravenous injection.

Mice were weighed regularly to monitor disease progression andeuthanized if their weight dropped lower than 75% of their initialweight.

Flow Cytometric Analysis of Peripheral Blood

Peripheral blood was isolated from mice by retro-orbital bleed at 41days post-transfer.

Total blood was stained with Biolegend's fluorescently labelledantibodies to CD45, CD11b, CD3, H2Kd (recipient) and H2Kb (donor) thenRBC were lysed using BD FACS Lysing solution (#349202). Cells werewashed once with PBS after lysis.

Labeled cells were run on the Miltenyi MACSquant and analyzed usingFlowJo.

Results

FIG. 49A-C illustrates the mean (A and B) weight loss and survival (c)for each treated group during the course of the experiment.Immune-suppressive α-human VISTA antibodies can be ranked based on theirimpact at suppressing or attenuating GVHD disease severity (weight loss)at the peak of disease (FIG. 49A).

INX904 (yellow) is poorly suppressive and only half the mice survive.INX901 (Green), INX902 (Red) and INX903 (orange) are stronglysuppressive and fully protective with INX901 completely inhibiting thedisease and INX902 and INX903 strongly attenuating disease severity. Asshown the α-human VISTA treated mice survive long term (FIGS. 49B andC).

After 41 days, peripheral blood is harvested from surviving mice andtested for chimerism (donor derived hematopoietic system) by stainingfor donor (H2Kb) or recipient (H2Kd) MHC class I. FIG. 50A illustratesone example of a surviving α-human VISTA treated mouse in which the vastmajority of CD11b in the blood express donor type MHC class I comparedto the blood of a BALB/c control. Of note, 4/8 mice that received bonemarrow cells only and 3/8 mice treated with INX901 failed to reachchimerism due to “failure of engraftment” (FIG. 50B) and wasretrospectively removed from the analysis. This phenomenon could beexplained by a suboptimal irradiation dose and the high potency ofINX901 to suppress T cell activation. It is well established that Tcells help engraftment especially at low doses of TBI (“The history andfuture of T-cell depletion as graft-versus-host disease prophylaxis forallogeneic hematopoietic stem cell transplantation”, Vincent T. Ho,Robert J. Soiffer Blood 2001 98:3192-3204;doi:10.1182/blood.V98.12.3192). Complete chimerism was achieved in 100%of the mice treated with INX902, INX903 and INX904. Donor derived Tcells were enumerated in the blood of all surviving chimeric mice. Allα-human VISTA treated surviving chimeric mice present a 4 to 9 timeshigher number of T cells in the blood than mice that received bonemarrow cells only (FIG. 50C) arguing against a depleting effect ofα-human VISTA antibodies.

Therefore, INX901, INX902, INX903 and INX904 α-human VISTA antibodiesall showed immune suppressive activities and partially attenuated orcompletely suppressed acute GvHD.

Example 28: Effects of α-Human VISTA Antibody INX901 (10 mg/kg) onXenogeneic GvHD in NSG Mice

Humanized mouse models of xenogeneic Graft-versus Host Disease (GvHD)allow the study of immunomodulatory compounds specific to human drugtargets in vivo. These are based on immune-deficient strains of miceinjected with peripheral blood mononuclear cells (PBMCs) from human. TheNOD-SCID IL-2Rγnull (NSG) strain lack mature T cells, B cells, andnatural killer cells and are amendable to xenogeneic GvHD studies. Inthe NSG model of xeno-GvHD, donor human T-cells expand robustly inrecipient mice and effect anti-host cell reactivity leading to cutaneoustissue infiltration. The mice lose weight and if left untreated willsuccumb to GvHD. The timeframe of disease progression can range from 3to 5 weeks. The time of disease occurrence and progression can beaccelerated by irradiating the mice with 3Gy prior to transfer of thehuman PBMCs. In this case the disease initiates after approximately 1-2weeks and mice will succumb by the 2-3-week mark.

Herein we describe the results of experiments wherein we examined theability of human VISTA specific antibody INX901 to modulate diseaseprogression in NSG GvHD. Briefly, we irradiated and injected the micewith 2.5 million human PBMCs along with a single dose of controlimmunoglobulin (Ig) or INX901. We confirmed the presence of humanT-cells by flow cytometry of peripheral blood taken from the recipientmice at 10 days post-treatment. Disease progression was monitored byregularly weighing mice. It was seen that NX901 drastically reduceddisease progress and increased mouse survival.

Materials and Methods

Mice and Disease Induction

7-week old NSG mice were purchased from the laboratory of Steve Fieringat DHMC and housed in SPF conditions at the DHMC. Mice were tattooedprior to the initiation of the experiment.

Human peripheral blood was isolated from apheresis cones provided from avolunteer donor at the DHMC blood donor program.

PBMCs were isolated by Ficoll gradient centrifugation.

Mice were irradiated with 3Gy prior to cellular transfer (DHMC).

Six mice received 2.5 million PBMCs diluted in 10 mg/ml human IgG (CrownBioscience, lot #AB150073) in 200 ul PBS. Six mice received 2.5 millionPBMCs diluted in 10 mg/ml INX901 (lot# BP-021-016-2) in 200 ul PBS. Alltherapeutics were delivered via tail vein injection.

Mice were weighed regularly to monitor disease progression andeuthanized if their weight dropped to lower than 80% of their startingweight.

Flow Cytometric Analysis of Peripheral Blood

Peripheral blood was isolated from mice by retro-orbital bleed at 10days post-treatment.

Cells were subjected to RBC lysis, thoroughly rinsed and stained withBiolegend's fluorescently labelled antibodies, anti-mouse CD45 BrV421,anti-mouse CD3-APC-Cy7, anti-human CD45-PE, and anti-human CD3-PE-Cy7using standard immunological flow cytometric protocols.

Labeled Cells were Run on the Miltenyi MACSquant and Analyzed UsingFlowJo.

Results

The experiment in FIG. 51A compares the mean weight of mice within thegroup during the course of the experiment. FIG. 51B compares the weightof individual mice. FIG. 52 shows the results of the flow cytometryanalysis of mouse peripheral blood at day 10 post-treatment. Theexperiment shows T-cell expansion in NSG mice treated with INX901 orcontrol AB in xeno-GvHD disease model where the values indicate the % oftotal CD45+ cells in the mouse peripheral circulation made up of humanCD3⁺ T-cells.

These results show that expansion of peripheral human T-cells is reducedin the INX901 treated group and that mice in the INX901-treated group donot lose weight as quickly or consistently as those in thecontrol-treated group. These results provide further evidence that VISTAagonist antibodies may be used to treat and prevent GvHD disease.

Example 29: Efficacy of Hamster α-Mouse VISTA Antibody 8G8 at 8 mg/Kg onGvHD in Irradiated BALB/c Mice Injected with C57/BI6 T Cells and BoneMarrow

We examined the immune-suppressive efficacy of Hamster α-mouse VISTAantibody 8G8 by its ability to modulate GVHD diseaseprogression/severity compared to a hamster IgG control.

Materials and Methods

10-week old males BALB/c recipient mice and C57/BI6 donor mice werepurchased from Jackson. All mice were housed in SPF conditions at theDHMC vivarium.

Recipient mice were subjected to total body irradiation (TBI) emanatingfrom a cesium-137 source at 300 cGy at Day-1 and D0 prior transfer.

Donor mice were euthanized and bone marrow was harvested by flushingfemur and tibia with HBSS. Red blood cells were lysed using ACKsolution. T cells were isolated from spleens by negative selection(Stemcell #19851).

Recipient mice received 10 million bone marrow cells and 2 million Tcells along with 200 ug Hamster IgG (BioXcell, BE0091 lot #18206/1015)or 8G8 (lot# AB130318-1). Cells and antibodies were administered by tailvein intravenous injection.

Mice were weighed regularly to monitor disease progression andeuthanized if their weight dropped lower than 75% of their initialweight.

Results

FIG. 53A-C illustrates the mean (a) and individual (b) weight loss andsurvival (c) for each treatment group during the course of theexperiment. Immune-suppressive Hamster α-mouse VISTA antibody 8G8strongly attenuates disease severity at the peak of disease (D8-10) andpromotes long-term survival in the majority of the mice. Specifically,FIG. 53A shows mean weight loss by group (N=8 mice per group); FIG. 53Bshows individual weight loss by group (N=8 mice per group) and FIG. 53Csurvival. These results show that 8G8 is immune-suppressive and stronglyattenuates acute GvHD severity promoting long term survival. Theseresults provide further evidence that VISTA agonist antibodies may beused to treat and prevent GvHD disease.

Example 30: Dose Efficacy of α-Human VISTA Antibodies INX902 (10, 2.5,and 1 mg/kg) on GvHD in Irradiated BALB/c Mice Injected with DDE1 TCells and Bone Marrow

In these experiments fully C57/B16 Human-VISTA Knock in mice (DDE1) wereused as donor for T cells and bone marrow transferred in irradiatedBALB/c recipient. Disease progression was monitored by regularlyweighting mice. We examined the immune-suppressive efficacy of theα-human VISTA antibody, INX902, at various doses compared to Human Igcontrol by measuring their ability to modulate diseaseprogression/severity. We also verified the presence of donor T-cells andchimerism in the surviving mice by flow cytometry of peripheral bloodtaken from the recipient mice at 21 days post-treatment.

Materials and Methods

GvHD Model and Disease Severity Evaluation

10-week old males BALB/c recipient mice were purchased from Jackson.14-weeks old males Human-KI VISTA donor mice (DDE1) on C57/B16background were purchased from Sage labs. All mice were housed in SPFconditions at the DHMC vivarium.

Recipient mice were subjected to total body irradiation (TBI) emanatingfrom a cesium-137 source at 350 cGy at Day−1 and D0 prior transfer.

Donor mice were euthanized and bone marrow was harvested by flushingfemur and tibia with HBSS. Red blood cells were lysed using ACKsolution. T cells were isolated from spleens by negative selection(Stemcell #19851).

Recipient mice received 10 million bone marrow cells and 2 million Tcells along with 10 mg/kg human IgG2 (Crown Bioscience, lot #AB150073)or 10 mg/kg, 2.5 mg/kg or 1 mg/kg INX902 (lot# BP-021-016-3). Cells andantibodies were administered by tail vein intravenous injection.

Mice were weighed regularly to monitor disease progression andeuthanized if their weight dropped lower than 75% of their initialweight.

Flow Cytometric Analysis of Peripheral Blood

Peripheral blood was isolated from mice by retro-orbital bleed at 21days post-transfer.

Total blood was stained with Biolegend's fluorescently labelledantibodies to CD45, CD11b, CD3, H2Kd (recipient) and H2Kb (donor) thenRBC were lysed using BD FACS Lysing solution (#349202). Cells werewashed once with PBS after lysis.

Labeled cells were run on the Miltenyi MACSquant® and analyzed usingFlowJo.

Results

Acute GvHD is induced in irradiated BALB/c recipient by transferringallogenic (C57/BI6) bone marrow and splenic T cells isolated from humanVISTA-KI mice. Disease severity is measured by following weight loss.

FIG. 54A-B illustrates the mean weight loss (A) and survival (B) forINX902 treated mice. INX902 attenuates disease severity at all dosestested as assessed by the reduced weight loss compared to Control Igtreated mice (FIG. 54A). INX902 appears more efficient at higher doses(10 m/kg) than at lower doses in preventing both weight loss (54A) andmortality (FIG. 54B).

After 21 days, peripheral blood is harvested from surviving mice andtested for chimerism (donor derived hematopoietic system) by stainingfor donor (H2Kb) or recipient (H2Kd) MHC class I. FIG. 55A illustratesthe percentage of chimerism in INX902 treated mice. With irradiationdoses of 350 cGy, all mice reached complete chimerism including thosethat received bone marrow cells only. FIG. 55B shows that T cell numbersare increased in INX902 treated mice compared to mice that received bonemarrow cells only. Specifically, FIG. 55A-B shows chimerism in survivingmice treated with various doses of INX902 or control Ig in acute GvHDdisease model. FIG. 55A shows the percentage of donor derived CD11b inthe blood of INX902 treated mice and FIG. 55A the donor derived T cellsnumber in 25 uL of blood in INX902 treated mice or in DDE1 control mice.

Therefore Acute GvHD is effectively treated in irradiated BALB/crecipient using the INX902 agonist antibody. These results providefurther evidence that VISTA agonist antibodies may be used to treat andprevent GvHD disease.

Example 31: Dose Efficacy of α-Human VISTA Antibodies INX901 and INX903(10, 2.5, and 1 Mg/Kg) on GvHD in Irradiated BALB/c Mice Injected withDDE1 T Cells and Bone Marrow

We examined the immune-suppressive efficacy of α-human VISTA antibodies,INX901 and INX903, at various doses compared to a human Ig control bymeasuring their ability to modulate GVHD disease progression/severity.We also verified chimerism in the surviving mice by flow cytometry ofperipheral blood taken from the recipient mice at 27-34 dayspost-treatment.

Materials and Methods

GvHD Model and Disease Severity Evaluation

9-week old males BALB/c recipient mice were purchased from Jackson.12-weeks old males Human-KI VISTA donor mice (DDE1) on C57/B16background were purchased from Sage labs. All mice were housed in SPFconditions at the DHMC vivarium.

Recipient mice were subjected to total body irradiation (TBI) emanatingfrom a cesium-137 source at 300 cGy at Day-1 and D0 prior transfer.

Donor mice were euthanized and bone marrow was harvested by flushingfemur and tibia with HBSS. Red blood cells were lysed using ACKsolution. T cells were isolated from spleens by negative selection(Stemcell #19851).

Recipient mice received 10 million bone marrow cells and 2 million Tcells along with 10 mg/kg human IgG2 (Crown Bioscience, lot #AB150073)or 10 mg/kg, 2.5 mg/kg or 1 mg/kg of INX901 (lot# BP-021-016-2) orINX903 (lot# BP-021-016-4). Cells and antibodies were administered bytail vein intravenous injection.

Mice were weighed regularly to monitor disease progression andeuthanized if their weight dropped lower than 75% of their initialweight.

Flow Cytometric Analysis of Peripheral Blood

Peripheral blood was isolated from mice by retro-orbital bleed at 27days (INX901) or 34 days (INX903) post-transfer.

Total blood was stained with Biolegend's fluorescently labelledantibodies to CD45, CD11b, CD3, H2Kd (recipient) and H2Kb (donor) thenRBC were lysed using BD FACS Lysing solution (#349202). Cells werewashed once with PBS after lysis.

Labeled cells were run on the Miltenyi MACSquant and analyzed usingFlowJo.

Results

Acute GvHD is induced in irradiated BALB/c recipient by transferringallogenic (C57/BI6) bone marrow and splenic T cells isolated from humanVISTA-KI mice. Disease severity is measured by following weight loss.FIG. 56A-D illustrates the mean (FIG. 56A and FIG. 56C) weight loss andsurvival (b and d) for INX901 (FIG. 56C and FIG. 56D) and INX903 (FIG.56A and FIG. 56B) treated mice during the course of the experiment.

INX903 attenuates disease severity at all doses tested as assessed bythe reduced weight loss compared to Control Ig treated mice (FIG. 56A).INX903 also increases survival at all dose tested with the lowest doseof 1 mg/kg appearing more protective than higher doses (FIG. 56B).

INX901 completely inhibits disease at all doses tested as assessed bythe absence of weight loss compared to Control Ig treated mice (FIG.56C). INX901 also increases survival at all doses tested with the lowestdose of 1 mg/kg appearing more protective than higher doses (FIG. 56D).

After 27 to 34 days, peripheral blood is harvested from surviving miceand tested for chimerism (donor derived hematopoietic system) bystaining for donor (H2Kb) or recipient (H2Kd) MHC class I.

FIG. 57A illustrates the percentage of chimerism in INX903 treated miceand FIG. 57B the percentage of chimerism in INX901 treated mice. Allmice that received bone marrow cells only and nearly all mice thatreceived T cells and were treated with INX901 failed to reach chimerismdue to “failure of engraftment” (FIG. 57A and FIG. 57B). This phenomenoncould be explained by a suboptimal irradiation dose and the high potencyof INX901 to suppress T cell activation. As noted above it is wellestablished that T cells help engraftment especially at low doses ofTBI. As evidence thereof, complete chimerism was achieved in almost allthe mice that received T cells and were treated with the lesssuppressive INX903 (FIG. 57A).

Therefore, INX901 and INX903 respectively suppress or attenuate acuteGvHD at doses as low as 1 mg/kg. Lower doses appear more effective thanhigher doses although mortality could also be due to engraftment failurein mice receiving strong immune suppressive antibodies in the context oflow doses of TBI. These results provide further evidence that VISTAagonist antibodies may be used to treat and prevent GvHD disease.

Example 32: Compared Efficacy of α-Human VISTA Antibodies INX803, INX804at 10 mg/Kg on GvHD in Irradiated BALB/c Mice Injected with DDE1 T Cellsand Bone Marrow

In these experiments we used fully C57/BI6 Human-VISTA Knock in mice(DDE1) as donor for T cells and bone marrow transferred in irradiatedBALB/c recipient. Disease progression was monitored by regularlyweighting mice. We examined the immune-suppressive efficacy of α-humanVISTA antibodies INX803 and INX804 compared to Human Ig control bymeasuring their ability to modulate disease progression/severity.

Materials and Methods

GvHD Model and Disease Severity Evaluation

9-week old males BALB/c recipient mice were purchased from Jackson.10-weeks old males Human-KI VISTA donor mice (DDE1) on C57/BI16background were purchased from Sage labs. All mice were housed in SPFconditions at the DHMC vivarium.

Recipient mice were subjected to total body irradiation (TBI) emanatingfrom a cesium-137 source at 350 cGy at Day−1 and D0 prior transfer.

Donor mice were euthanized and bone marrow was harvested by flushingfemur and tibia with HBSS. Red blood cells were lysed using ACKsolution. T cells were isolated from spleens by negative selection(Stemcell #19851).

Recipient mice received 10 million bone marrow cells and 2 million Tcells along with 10 mg/kg human IgG2 (Crown Bioscience, lot #AB150073)or INX803 (lot# BP-018-016), INX804 (lot# BP-019-016). Cells andantibodies were administered by tail vein intravenous injection.

Mice were weighed regularly to monitor disease progression andeuthanized if their weight dropped lower than 75% of their initialweight.

Results

FIG. 58A-C illustrates the mean (58A) and individual (588) weight lossand survival (58C) for each treated group during the course of theexperiment. INX803 (Green) is strongly suppressive, inhibits diseasedevelopment and fully protects the mice. INX804 attenuates diseaseseverity at the peak of disease but fails to provide complete protectionto the mice long term, half of which eventually succumb to GvHD.Therefore, the tested α-human VISTA antibodies showed immune-suppressiveactivity in their ability to attenuate or completely suppress acuteGvHD. These results provide further evidence that anti-human VISTAagonist antibodies may be used to treat and prevent GvHD disease.

Example 33: Efficacy of α-Human VISTA Antibodies INX800 and INX801 onGvHD in Irradiated BALB/c Mice Injected with DDE1 T Cells and BoneMarrow

In these experiments we again used fully C57/BI6 Human-VISTA Knock inmice (DDE1) as donor for T cells and bone marrow transferred inirradiated BALB/c recipient. Disease progression was monitored byregularly weighing mice. We examined the immune-suppressive efficacy ofα-human VISTA antibodies INX800 and INX801, at 10 mg/kg compared toHuman Ig control by measuring the ability to modulate diseaseprogression/severity.

Materials and Methods

GvHD Model and Disease Severity Evaluation

9-week old males BALB/c recipient mice were purchased from Jackson.12-weeks old males Human-KI VISTA donor mice (DDE1) on C57/B16background were purchased from Sage labs. All mice were housed in SPFconditions at the DHMC vivarium.

Recipient mice were subjected to total body irradiation (TBI) emanatingfrom a cesium-137 source at 300 cGy at Day−1 and D0 prior transfer.

Donor mice were euthanized and bone marrow was harvested by flushingfemur and tibia with HBSS. Red blood cells were lysed using ACKsolution. T cells were isolated from spleens by negative selection(Stemcell #19851).

Recipient mice received 5 million bone marrow cells and 1.6 million Tcells along with 10 mg/kg human IgG2 (Crown Bioscience, lot #AB150073)10 mg/kg of INX800 or INX801. Cells and antibodies were administered bytail vein intravenous injection.

Mice were weighed regularly to monitor disease progression andeuthanized if their weight dropped lower than 80% of their initialweight.

Results

Both INX800 and INX801 mice lost less weight than the control treatedgroup (FIG. 59). In fact, while all mice from the control group had tobe sacrificed within 2 weeks, all of the mice treated with INX800 orINX801 survived for >32 days (FIG. 59). In the experiment acute GvHD wasinduced by transfer of T cells and BM from hV-KI mice into irradiatedBalb/c recipients. Mice were tracked for disease by weight loss, withmice being sacrificed if more than 20% of the initial starting weightwas lost.

Therefore, both INX800 and INX801 attenuate acute GvHD at a dose of 10mg/kg. These mice suffered less weight loss and showed increasedsurvival over the Ig control group. These results provide furtherevidence that anti-human VISTA agonist antibodies may be used to treatand prevent GvHD disease, both acute and chronic forms.

Example 34: Effects of Anti-Murine Vista Antibodies in NZBWF-1 LupusModel

NZBWF-1 Lupus Model

New Zealand black x New Zealand white (NZBWF-1) is a commerciallyavailable lupus prone strain available through The Jackson Laboratory.These mice spontaneous develop lupus similar to systemic lupuserythematosus (SLE) patients with prevalence in female mice. Hallmarksof disease include the onset of proteinuria, glomerulonephritis,elevated levels of self-reactive antibodies such as ds DNA antibodies,hemolytic anemia and immune complex deposition in the kidneys. At thecellular level, T cell, B cell and myeloid cell abnormalities have beenreported.

In these experiments, we examined the function of 8G8, a hamster α mouseVISTA antibody in female NZBWF-1 mice. Mice were treated three times aweek with control-Ig or 8G8. Mice were monitored weekly for proteinuriaand body weight. Serum was collected every two weeks during thetreatment. At the end of the experiment, serum, spleens and kidneys wereharvested. Serum was stored at −80° C. until required for Luminex assay.

Spleens were processed for flow cytometric analysis, cell sorting orsnap frozen in OCT for immunofluorescence staining and RNA isolation forgene profiling and nanoString analysis. One kidney was snap frozen inOCT for immunofluorescence staining and RNA isolation for gene profilingand nanoString analysis. The second kidney was fixed and paraffinembedded. Paraffin sections were H&E stained for clinical pathology.

Materials and Methods

Mouse Treatment

8-week old female NZBWF-1 mice were purchased from Jackson and stored inSPF conditions at the DHMC.

Proteinuria and body weight were monitored weekly in the DartmouthHitchcock animal facility.

Control-IgG/Hamster Ig or 8G8 was administered three times a week at 300μg/mouse by i.p injection.

Mice were sacrificed upon signs of poor health and reduced activity, andaccording to animal facility protocols.

Proteinuria

Chemstrips 10 were purchased from Roche. Urine was collected from miceand placed onto the chemstrip. To determine protein in the urine, thecolorimetric scale was used: 0 mg/dL, trace (1 mg/dL), 30 mg/dL, 100mg/dL and 500 mg/dL.

Serum Analysis

Serum was collected and stored at −80° C. until required. Chemokine andcytokine levels were determined using a 32 Milliplex MouseCytokine/Chemokine Magnetic Bead Panel (Millipore) and the assay run ona Bio-plex 200 System (Life Science Research, Bio Rad). Data wasanalyzed using the Bio-Plex Manager 6.0 software.

Clinical Pathology

Kidneys were placed into cassettes and fixed O/N in 10% Formalin at roomtemperature, then briefly washed in PBS and transferred and kept into70% Ethanol (Fisher Scientific) prior to being transferred to thePathology Translational Research Core at the Geisel School of Medicineat Dartmouth where they were paraffin embedded, sectioned and thenstained.

Paraffin embedded tissue sections (4 μm) were stained using a Leica BONDRX automated stainer. After dewaxing, the sections were subjected toantigen retrieval (Bond epitope retrieval solution 2, 100° C., 20 min)and incubated with the primary antibody (see dilution below) for 30-60min, at room temperature in Leica diluent. Slides are then washed 3×5min washes in PBS and incubated with secondary antibody (from Leica BondRefine detection kit, DS9800). After 3 final washes in PBS the sectionswere incubated with DAB (Leica Bond polymer detection kit), rinsed,counterstained with hematoxylin and mounted.

Clinical pathology will be assessed by a pathologist.

Results

This experiment was designed to examine whether 8G8 mediated animmunosuppressive role in female NZBWF-1 mice. Mice were monitoredweekly from 16 weeks old for proteinuria development. At week 32, theweek after proteinuria was detected, mice were treated with 300 μg ofHamster Ig or 8G8 by i.p injection three times a week. At week 33 allmice in the Hamster Ig group were sacrificed due to poor health, andspleens, kidneys and plasma were collected. AS shown in FIG. 60, themice in the 8G8 group displayed better health and reduced proteinuria.

To determine the effect of 8G8 on mediators in the plasma, a 32Multiplex Mouse Cytokine/Chemokine Magnetic Luminex assay was run. Asignificant reduction in LIX/CXCL5 and an increase in IL-9 was detected(FIG. 61). Specifically, as shown in FIG. 61 LIX/CXCL5 and IL-9 levelsin the serum of Control-Ig and 8G8 treated NZBWF-1 mice were detected.Serum was collected at week 33 from Control-IgG (n=5) and 8G8 mice (n=5)and chemokines and cytokines were assessed on a 32 plex run usingBio-plex 200 Systems and analyzed by Bio Plex manager 6.0 software. Datais shown as the mean+/−SEM and statistical significance was determinedby the unpaired Student t Test. In FIG. 61 **denotes significance(p<0.01) between groups.

The decrease in LIX/CXCL5 is of note as its expression is regulated byIL-17 and is a pathogenic cytokine in lupus. It is also associated withneutrophil recruitment and accelerated atherosclerosis in SLE(Nalbandian et al., “Interleukin-17 and systemic lupus erythematosus:current concepts”, Clinical and Experimental Immunology. 2009;157(2):209-15; Lopez-Ped rera et al., “Accelerated atherosclerosis insystemic lupus erythematosus: role of proinflammatory cytokines andtherapeutic approaches”, Journal of Biomedicine & Biotechnology, 2010Article ID 607084). The increase of IL-9 suggests 8G8 promotes andanti-inflammatory environment, as IL-9 can play a role in reducinginflammation in SLE (Leng et al., “Potential roles of IL-9 in thepathogenesis of systemic lupus erythematosus”, American Journal ofClinical and Experimental Immunology 2012; 1(1):28-32)

Conclusion

The agonistic anti-VISTA agonistic 8G8 improved survival, increasedprotective anti-inflammatory cytokines and reduced inflammatorycytokines and further reduced the development of proteinuria (FIG. 60,61). These results indicate that agonistic anti-VISTA antibodies may beused in the treatment or prevention of lupus and for managing thepathological side effects of lupus such as its deleterious effects onkidney function and for enhancing survival.

Example 35: Effects of Anti-Mouse Vista Antibody in NZBWF-1 Lupus Model

NZBWF-1 Lupus Model

The NZBWF-1 lupus model is described supra. In this group of experimentsusing the NZBWF-1 lupus model, we again examined the function of 8G8, ahamster α mouse VISTA antibody in female NZBWF-1 mice. Mice were treatedthree times a week with control-Ig or 8G8. Mice were monitored weeklyfor proteinuria and body weight. Serum was collected every two weeksduring the treatment. At the end of the experiment, serum, spleens andkidneys were harvested. Serum was stored at −80° C. until required forLuminex assay.

Spleens were processed for flow cytometric analysis, cell sorting orsnap frozen in OCT for immunofluorescence staining and RNA isolation forgene profiling and nanoString analysis. One kidney was snap frozen inOCT for immunofluorescence staining and RNA isolation for gene profilingand nanoString analysis. The second kidney was fixed and paraffinembedded. Paraffin sections were H&E stained for clinical pathology.

Materials and Methods

Mouse Treatment

8-week old female NZBWF-1 mice were purchased from Jackson and stored inSPF conditions at the DHMC.

Proteinuria and body weight were monitored weekly in the DartmouthHitchcock animal facility.

Control-IgG/Hamster Ig or 8G8 was administered three times a week at 300μg/mouse by i.p injection

Mice were sacrificed upon signs of poor health and reduced activity, andaccording to animal facility protocols.

Proteinuria

Chemstrips 10 were purchased from Roche. Urine was collected from miceand placed onto the chemstrip. To determine protein in the urine, thecolorimetric scale was used: 0 mg/dL, trace (1 mg/dL), 30 mg/dL, 100mg/dL and 500 mg/dL.

Serum Analysis

Serum was collected and stored at −80° C. until required. Chemokine andcytokine levels were determined using a 32 Milliplex MouseCytokine/Chemokine Magnetic Bead Panel (Millipore) and the assay run ona Bio-plex 200 System (Life Science Research, Bio Rad). Data wasanalyzed using the Bio-Plex Manager 6.0 software.

Myeloid-Derived Suppressor Cell Isolation Kit

Myeloid-Derived Suppressor Cells (MDSCs) were isolated using theMyeloid-Derived Suppressor Cell Isolation Kit from Miltenyi Biotecaccording to the manufacturer's instructions.

RNA Isolation and nanoString

RNA was isolated from MDSCs using Trizol (Life Technologies) and thePureLink RNA Mini Kit (Ambion). RNA was run on a mouse inflammatorynanoString 12 assay (nanoString Technologies) and the data wasquantified using the nSolver Analysis Software.

Clinical Pathology

Kidneys were placed into cassettes and fixed O/N in 10% Formalin at roomtemperature, then briefly washed in PBS and transferred and kept into70% Ethanol (Fisher Scientific) prior to being transferred to thePathology Translational Research Core at the Geisel School of Medicineat Dartmouth where they were paraffin embedded, sectioned and thenstained.

Paraffin embedded tissue sections (4 μm) were stained using a Leica BONDRX automated stainer. After dewaxing, the sections were subjected toantigen retrieval (Bond epitope retrieval solution 2, 100° C., 20 min)and incubated with the primary antibody (see dilution below) for 30-60min, at room temperature in Leica diluent. Slides are then washed 3×5min washes in PBS and incubated with secondary antibody (from Leica BondRefine detection kit, DS9800). After 3 final washes in PBS the sectionswere incubated with DAB (Leica Bond polymer detection kit), rinsed,counterstained with hematoxylin and mounted.

Clinical pathology will be assessed by a pathologist.

Results

This experiment was designed to examine whether 8G8 mediated animmunosuppressive role in female NZBWF-1 mice. Mice were monitoredweekly from 22 weeks old for proteinuria development. On week 28, theweek after proteinuria was detected; mice were treated with 300 μg ofHamster Ig or 8G8 by i.p injection three times a week. Whereas diseaseseverity in the control group continued to increase, the mice in the 8G8group displayed reduced proteinuria levels (FIG. 62).

As shown in the figure the agonistic anti-VISTA antibody 8G8 reduced thedevelopment of proteinuria (FIG. 62).

Example 36: Evaluation of Anti-Vista Antibodies in I MRL/Ipr Lupus Model

I MRL/Ipr Lupus Animal Model

As noted, MRL/Ipr is a commercially available lupus prone strainavailable through The Jackson Laboratory. These mice show signs oflymphoproliferation due to a spontaneous mutation in Fas (FasIpr).Hallmarks of disease include immune complex glomerulonephrosis and highlevels of circulating immune complexes. Abnormalities in the T cellcompartment have also been reported. In this experiment, we examined thefunction of 8G8, a hamster α mouse VISTA antibody in female MRL/Iprmice. Mice were treated three times a week with control-Ig/hamster-Ig or8G8. Mice were monitored weekly for proteinuria and body weight. Serumwas collected every two weeks during the treatment. At the end of theexperiment, serum, spleens and kidneys were harvested. Serum was storedat −80° C. until required for Luminex assay. Spleens and lymph nodeswere processed for cell sorting or snap frozen in OCT forimmunofluorescence staining and RNA isolation for gene profiling andnanoString analysis. One kidney was snap frozen in OCT forimmunofluorescence staining and RNA isolation for gene profiling andnanoString analysis. The second kidney was fixed and paraffin embedded.Paraffin sections were H&E stained for clinical pathology.

Materials and Methods

Mouse Treatment

12-week old female NZBWF-1 mice were purchased from Jackson and storedin SPF conditions at the DHMC.

Proteinuria and body weight was monitored weekly in the DartmouthHitchcock animal facility.

Control-Ig/Hamster Ig or 8G8 was administered three times a week at 300μg/mouse by i.p injection

Mice were sacrificed when proteinuria was 500 mg/dL.

Proteinuria

Chemstrips 10 were purchased from Roche. Urine was collected from miceand placed onto the chemstrip. To determine protein in the urine, thecolorimetric scale was used: 0 mg/dL, trace (1 mg/dL), 30 mg/dL, 100mg/dL and 500 mg/dL.

Serum Analysis

Serum was collected and stored at −80° C. until required. Chemokine andcytokine levels were determined using a 32 Milliplex MouseCytokine/Chemokine Magnetic Bead Panel (Millipore) and the assay run ona Bio-plex 200 System (Life Science Research, Bio Rad). Data wasanalyzed using the Bio-Plex Manager 6.0 software.

RNA Isolation and nanoString

RNA was isolated using Trizol (Life Technologies) and the PureLink RNAMini Kit (Ambion). RNA was run on a mouse inflammatory nanoString 12assay (nanoString Technologies) and the data was quantified using thenSolver Analysis Software.

Clinical Pathology

Kidneys were placed into cassettes and fixed O/N in 10% Formalin at roomtemperature, then briefly washed in PBS and transferred and kept into70% Ethanol (Fisher Scientific) prior to being transferred to thePathology Translational Research Core at the Geisel School of Medicineat Dartmouth where they were paraffin embedded, sectioned and thenstained.

Paraffin embedded tissue sections (4 μm) were stained using a Leica BONDRX automated stainer. After dewaxing, the sections were subjected toantigen retrieval (Bond epitope retrieval solution 2, 100° C., 20 min)and incubated with the primary antibody (see dilution below) for 30-60min, at room temperature in Leica diluent. Slides are then washed 3×5min washes in PBS and incubated with secondary antibody (from Leica BondRefine detection kit, DS9800). After 3 final washes in PBS the sectionswere incubated with DAB (Leica Bond polymer detection kit), rinsed,counterstained with hematoxylin and mounted.

Clinical pathology will be assessed by a pathologist.

Results

This experiment was designed to examine whether 8G8 mediated animmunosuppressive role in female MRL/pr mice. Mice were monitored weeklyfrom 15 weeks old for proteinuria development. On week 16, mice weretreated with 300 μg of Hamster Ig or 8G8 by i.p injection three times aweek. To determine the effect of 8G8 on mediators in the plasma wascollected and stored at −80° C. Spleens and lymph nodes were harvestedand snap frozen for immunofluorescence staining and RNA isolation. Theagonistic anti-VISTA antibody 8G8 reduced the incidence of diseasedevelopment as characterized by high proteinuria levels (see FIG. 63).

As shown in FIG. 63 the agonist anti-mouse VISTA antibody 8G8 reducesproteinuria development in MRL/Ipr mice in an experiment wherein 15 weekold female MRL/pr mice were monitored weekly for proteinuria.Proteinuria values were recorded using chemstrips and quantified asmg/dL. On week 16, mice were treated with either 300 ug hamster-Ig(black line, n=8) or 300 ug 8G8 (red line, n=8) by i.p injection threetimes a week. Data at week 21 were discarded due to technical problemswith the chemstrips. (FIG. 63 A) Average proteinuria is shown withstandard error bars. (FIG. 63 B) Disease incidence at each time pointwas calculated as the percent of mice in each group that exhibitedproteinuria at or greater than 100 mg/dL. Accordingly 8G8 reduced theincidence of disease development as characterized by high proteinurialevels.

As shown in FIG. 64 8G8 also reduces splenomegaly in MRL/Ipr mice in anexperiments wherein spleens were harvested on week 23 from mice weretreated with either 300 ug Control-Ig/hamster-Ig or 300 ug 8G8 by i.pinjection three times a week. Splenomegaly was observed in Control-Igtreated mice compared to 8G8 treated mice. (representative spleens shownin the Figure).

As further shown in FIG. 65 8G8 also reduces lymphoproliferation ofcervical lymph nodes in MRL/Ipr mice in experiments wherein cervicallymph nodes were harvested on week 23 from mice treated with either 300ug Control-Ig/hamster-Ig or 300 ug 8G8 by i.p injection three times aweek. Lymphoproliferation was observed in Control-Ig treated micecompared to 8G8 treated mice. Shown here are representative cervicallymph nodes.

These results further indicate that agonistic anti-VISTA antibodies maybe used in the treatment or prevention of lupus and for managing thepathological side effects of lupus such as its deleterious effects onkidney function, and on the spleen and on pathologicallymphoproliferation.

Example 37: Evaluation of Anti-Vista Antibodies in I MRL/Ipr Lupus Model

I MRL/Ipr Lupus Animal Model

The MRL/Ipr lupus model is described supra. In this experiment, we againexamined the effects of 8G8, a hamster α mouse VISTA antibody in femaleMRL/Ipr mice. Mice were treated three times a week with PBS,control-Ig/hamster-Ig or 8G8. Mice were monitored weekly for proteinuriaand body weight. Serum was collected every two weeks during thetreatment. At the end of the experiment, serum, lymph nodes, spleens andkidneys were harvested. Serum was stored at −80° C. Organs were fixedand paraffin embedded. Paraffin sections were H&E stained for clinicalpathology.

Materials and Methods

Mouse Treatment

8-week old female MRL/Ipr mice were purchased from Jackson and stored inSPF conditions at the DHMC.

Proteinuria and body weight was monitored weekly in the DartmouthHitchcock animal facility.

10 mg/kg/mouse control-Ig/hamster Ig or 8G8 or 200 uL of PBS wasadministered three times a week by i.p injection starting at week 11.

Mice were sacrificed when proteinuria was 500 mg/dL.

Proteinuria

Chemstrips 10 were purchased from Roche. Urine was collected from miceand placed onto the chemstrip. To determine protein in the urine, thecolorimetric scale was used: 0 mg/dL, trace (1 mg/dL), 30 mg/dL, 100mg/dL and 500 mg/dL.

Serum Analysis

Serum was collected and stored at −80° C. until required.

Clinical Pathology

Kidneys were placed into cassettes and fixed O/N in 10% Formalin at roomtemperature, then briefly washed in PBS and transferred and kept into70% Ethanol (Fisher Scientific) prior to being transferred to thePathology Translational Research Core at the Geisel School of Medicineat Dartmouth where they were paraffin embedded, sectioned and thenstained.

Paraffin embedded tissue sections (4 μm) were stained using a Leica BONDRX automated stainer. After dewaxing, the sections were subjected toantigen retrieval (Bond epitope retrieval solution 2, 100° C., 20 min)and incubated with the primary antibody (see dilution below) for 30-60min, at room temperature in Leica diluent. Slides are then washed 3×5min washes in PBS and incubated with secondary antibody (from Leica BondRefine detection kit, DS9800). After 3 final washes in PBS the sectionswere incubated with DAB (Leica Bond polymer detection kit), rinsed,counterstained with hematoxylin and mounted.

Clinical pathology will be assessed by a pathologist.

Results

This experiment was designed to examine whether 8G8 mediated animmunosuppressive role in female MRL/Ipr mice. Mice were monitoredweekly from 9 weeks old for proteinuria development. On week 11, micewere treated with 200 uL of PBS or 10 mg/kg of hamster Ig or 8G8 by i.pinjection three times a week. To determine the effect of 8G8 onmediators in the plasma was collected and stored at −80° C.

Thus, the VISTA agonist 8G8 reduced the incidence of disease developmentas characterized by high proteinuria levels (see FIG. 66). In theexperiments 9 week old female MRL/Ipr mice were monitored weekly forproteinuria. Proteinuria values were recorded using chemstrips andquantified as mg/dL. On week 11, mice were treated with either 200 uLPBS (dotted black line, n=8) or 10 mg/kg hamster-Ig (solid black line,n=8) or 10 mg/kg 8G8 (red line, n=8) by i.p injection three times aweek. (FIG. 66A) Average proteinuria is shown with standard error bars.(FIG. 66B) Disease incidence at each time point was calculated as thepercent of mice in each group that exhibited proteinuria at or greaterthan 100 mg/dL.

These results further indicate that agonistic anti-VISTA antibodies maybe used in the treatment or prevention of lupus and for managing thepathological side effects of lupus such as its deleterious effects onkidney function.

Example 38: Effects of Anti-Human VISTA Agonist Antibody INX903 onDisease Induction in a Mouse Model of Systemic Lupus Erythematosus

In the present experiment, we tested the effects of an anti-human VISTAagonist antibody in an accepted lupus model. In this murine model SLE isinduced by the transfer of human VISTA knock-in DDE1 CD8 depletedsplenocytes (donor) into a B6D2F1 host (recipient) Further, in thismodel, donor CD4 T cell polyclonal activation drives cognate host B cellactivation, expansion, and their production of autoantibodies leading torenal disease.

This lupus animal model meets 4 out of 11 American College ofRheumatology (ACR) criteria for lupus (“Systemic lupus erythematosus;”Tsokos GC. N Engl J Med. 2011 Dec. 1; 365(22):2110-21). Lupus-likefeatures of B6 CD8 depleted transferred to B6D2F1 model include: (1)Immune complex glomerulonephritis; (2) anti-nuclear ab; (3) anti-dsDNAab; and (4) anti-RBC ab (Coombs positivity). Additionally, this modelmeets sex-based differences in renal disease severity.

More particularly, we tested the effects of INX903 (human anti-humanVISTA-IgG2 wherein treatment was effected in the early stages of SLEinduction. It is accepted in the art that two-week phenotypes reliablypredict the long-term clinical phenotypes and can serve as earlysurrogate markers for long-term disease (“Advances in lupus stemmingfrom the parent-into-F1 model”. Via CS. Trends Immunol., 2010 Jun.31(6):236-45).

Materials and Methods

Study Design

A schematic summarizing the experimental parameters is contained in FIG.67. As shown therein INX903 (anti-human IgG2 agonist antibody containingvariable regions of VSTB95 antibody) was administered at days 0, 2, and6 following DDE1 transfer. At each time point, 4 mice per group wereanalyzed plus 1 naïve mouse. Spleens were processed for flow cytometry,and serum was recovered from cardiac blood for detection of anti-dsDNAIgG by ELISA.

Mice

Human VISTA KI (DDE1) mice have the human VISTA cDNA knocked-in in placeof the mouse VISTA gene, and express only human VISTA both at RNA andprotein level. The mice are bred at Sage Labs (Boyertown, Pa.). Themice, aged 8-12 weeks, first transited for 3 weeks in the quarantinefacility, and then were transferred to our regular facility. 4-month oldfemale DDE1 mice were used. 9 weeks old female B6D2F1 mice werepurchased from the Jackson Lab.

Spleen Cell Isolation and Transfer

The equivalent of 1 DDE1 donor spleen was transferred per B6D2F1 mouserecipient.

Single cell suspensions were prepared from 36 DDE1 spleens by mechanicaldisruption. RBCs were lysed with ACK.

CD8 T cells were depleted using Dynabeads® Mouse CD8 (Lyt 2)(Thermofisher #11447D) according to manufacturer instruction.

To follow cell proliferation, spleen cells were stained with Cell TraceViolet (Cell Trace™ Violet Cell Proliferation Kit, for flow cytometry(Thermofisher # C34557) according to manufacturer instruction.

A total of 1,230×10⁶ CD8 depleted spleen cells was obtained.

Each B6D2F1 mouse received 34×10⁶ CD8 depleted spleen cells in a volumeof 200 μl per tail vein injection (iv).

Anti-Human VISTA Antibodies and Dosage

INX903 was dosed at 10 mg/kg.

At day 0, antibodies were directly added to the cell suspension justprior IV transfer into recipient B6D2F1 mice.

Mice were then dosed on day 2 and 6 via intraperitoneal (ip) injections.

Control group received human IgG2 (Lot AB150073—4.7 mg/mL).

Treated group received INX903 (Lot BP-021-016-4—6 mg/mL).

Analysis of Immune Cell by Flow

At day 1, 3, 7 and 14 after donor cell transfer, donor CD4 T cellactivation, proliferation, accumulation, and host B cell activation andaccumulation were evaluated by flow cytometry on the spleens. Fouranimals per group+1 naïve mouse were analyzed at each time point.Spleens were mechanically disrupted, RBCs were lysed using ACK bufferand total viable nucleated cell number was evaluated using a Cellometerautomated cell counting system and AOPI. 2 million cells were stainedwith the following panels as shown below: Day 1

B3 B4 R2 B1 B2 PerCp PE- R1 APC- V1 V2 FITC PE Cy5.5 Cy7 AF647 Cy7 BV421BV510 IAd H2Kd B220 CD69 H2Kb CD4 CTV Yellow FcBlock LD 1/ 1/200 1/2001/200 1/200 1/300 NA 1/ 1/200 1000 1000

Following days:

B3 B1 B2 PerCp B4 R1 R2 V1 V2 FITC PE Cy5.5 PE-Cy7 AF647 APC-Cy7 BV421BV510 IAd H2Kd B220 CD25 H2Kb CD4 CTV Yellow FcBlock LD 1/1000 1/2001/200 1/200 1/200 1/300 NA 1/1000 1/200

Samples were run on a MacsQuant flow cytometer and analyzed with theFlowJo program.

Donor CD4 T cells were identified as Live, CD4+B220− H2Kb+ H2Kd− andanalyzed for activation marker expression (CD69 or CD25) andproliferation (Cell Trace Violet dilution). Donor CD4 cell number perspleen were calculated by applying the percentage of donor CD4 T cellson total viable cell number in the spleens.

Recipient B cells were identified as Live, CD4-B220+ H2Kb+ H2Kd+ andanalyzed for MHC class II expression. At each time point a naïve B6D2F1served for basal MHC class II expression comparison. Recipient B cellnumbers per spleen were calculated by applying the percentage of Host Bcells on total viable cell number in the spleens.

Anti-Double Stranded DNA Detection

Anti-dsDNA IgG in the serum at day 7 and 14 were quantified using anELISA kit from Alpha Diagnostic (Cat. No. 5120) according tomanufacturer instruction.

Identification of Recipient and Donor Cell Population

As shown in FIG. 68, the SLE mice generated by transfer of DDE1 spleencells (minus CD8⁺ cells) show the presence of both donor and recipient Band CD4 T cells. Donor CD4 T cells were identified as Live, CD4⁺B220H2Kb⁺ H2Kd. Recipient B cells were identified as Live, CD4 B22⁺ H2Kb+H2K⁺. In the experiments in FIG. 68 donor and host cell populations aredistinguished by their MHC class I alleles. The host B6D2F1 cellsexpress both H-2Kb and H-2Kd, whereas the donor DDE1 cells express onlyH-2Kb.

INX903 Leads to Decreases in SLE Disease Progression

To determine if the anti-VISTA agonist antibody INX903 can affect earlydisease progression, we evaluated recipient B cell activation,production of dsDNA antibodies, CD4 T cell activation and proliferation.It was shown that INX903 administration leads to a decrease in recipientB cell activation and accumulation and resulting splenomegaly. B cellactivation was demonstrated by the increased expression of MHC class III^(Ad) over time following the transplantation in the human IgG2(HuIgG2) treated group while no change in IAd was noted in the INX903treated group which appear similar to naïve mice. These results areshown in FIG. 69A. To note, this is an indirect effect as B cells fromrecipient mice do not express human VISTA and as such cannot responddirectly to INX903.

B cell gradual expansion following the transplantation in the human IgG2(HuIgG2) treated group reaching 200% of normal F1 values at D14 andresulting in mild splenomegaly is also prevented in the INX903 treatedgroup (See FIGS. 69B and C). The data in the figure further show that Bcells activation during SLE progression is also prevented by INX903treatment. FIG. 69A contains histogram plots of MHCII IAd expression onrecipient B cells. FIG. 69B shows the total number of recipient B cellsand spleen cells over the course of the experiment and MHC class II IAdMFI on recipient B cell over the course of the experiment (n=4, SEM).FIG. 69C shows spleen size in the treated animal at D14.

The experimental data in FIG. 70 further demonstrates that INX903administration leads to reduced anti-dsDNA autoantibodies production. Inthese experiments anti-dsDNA IgG titer in serum measured by ELISA innaïve (n=2), and HuIgG2 or INX903 treated mice at D7 and D14 (n=4, SEM).The experimental data in FIG. 71 additionally shows that INX903administration leads to a decrease in T cell activation andproliferation. As shown therein CD69 expression is decreased at earlytime points. Specifically it can be seen that CD69 expression isdecreased at Day 1 in INX903-treated CD4 T cells (n=4).

The experimental data in FIGS. 72 and 73 further shows that there is asustained reduction in the accumulation of donor CD4+ T cells followingtransfer. Despite dividing with the same frequency as the huIgG2-treatedgroup CD4 T cells in INX903-treated mice are dramatically decreased overtime (n=4). These experimental results show that administration of theexemplified anti-human VISTA agonist Ab resulted in the following: (i)reduced T cell proliferation and activation (this is the only modelavailable where disease-initiating T cells can be tracked); (ii) reducedcognate B cell activation (MHCII expression) and accumulation; (iii)reduced splenomegaly and (iv) reduced anti-dsDNA IgG auto-antibodiesproduction.

While observed after a relatively short duration, these results aresignificant because in this model, 2-week phenotypes are known toreliably predict the long-term clinical phenotypes and can serve asearly surrogate markers for long-term disease.

Example 39: Effects of the α-Mouse VISTA Antibody 8G8 on Systemic LupusErythematosus—MRL/Ipr—SCD8G8MRL1

In this experiment, we again examined the effects of 8G8, a hamsteranti-mouse VISTA agonist antibody in female MRL/Ipr mice. Mice weretreated three times a week with control-Ig/hamster-Ig or 8G8. Mice weremonitored weekly for proteinuria and body weight. Serum was collectedevery two weeks during the treatment. At the end of the experiment,serum, spleens and kidneys were harvested. Serum was stored at −80° C.until required for luminex assay. Spleens and lymph nodes were processedfor cell sorting or snap frozen in OCT for immunofluorescence stainingand RNA isolation for gene profiling and nanoString analysis. One kidneywas snap frozen in OCT for immunofluorescence staining and RNA isolationfor gene profiling and nanoString analysis. The second kidney was fixedand paraffin embedded. Paraffin sections were H&E stained for clinicalpathology.

Materials and Methods

Mouse Treatment

12-week old female NZBWF-1 mice were purchased from Jackson and storedin SPF conditions at the DHMC.

Proteinuria and body weight was monitored weekly in the DartmouthHitchcock animal facility.

Control-Ig/Hamster Ig or 8G8 was administered three times a week at 300μg/mouse by i.p injection

Mice were sacrificed when proteinuria was 500 mg/dL.

Proteinuria

Chemstrips 10 were purchased from Roche. Urine was collected from miceand placed onto the chemstrip. To determine protein in the urine, thecolorimetric scale was used: 0 mg/dL, trace (1 mg/dL), 30 mg/dL, 100mg/dL and 500 mg/dL.

Serum Analysis

Serum was collected and stored at −80° C. until required. Chemokine andcytokine levels were determined using a 32 Milliplex MouseCytokine/Chemokine Magnetic Bead Panel (Millipore) and the assay run ona Bio-plex 200 System (Life Science Research, Bio Rad). Data wasanalyzed using the Bio-Plex Manager 6.0 software.

RNA Isolation and nanoString

RNA was isolated using Trizol (Life Technologies) and the PureLink RNAMini Kit (Ambion). RNA was run on a mouse inflammatory nanoString 12assay (nanoString Technologies) and the data was quantified using thenSolver Analysis Software.

Clinical Pathology

Kidneys were placed into cassettes and fixed O/N in 10% Formalin at roomtemperature, then briefly washed in PBS and transferred and kept into70% Ethanol (Fisher Scientific) prior to being transferred to thePathology Translational Research Core at the Geisel School of Medicineat Dartmouth where they were paraffin embedded, sectioned and thenstained.

Paraffin embedded tissue sections (4 μm) were stained using a Leica BONDRX automated stainer. After dewaxing, the sections were subjected toantigen retrieval (Bond epitope retrieval solution 2, 100° C., 20 min)and incubated with the primary antibody (see dilution below) for 30-60min, at room temperature in Leica diluent. Slides are then washed 3×5min washes in PBS and incubated with secondary antibody (from Leica BondRefine detection kit, DS9800). After 3 final washes in PBS the sectionswere incubated with DAB (Leica Bond polymer detection kit), rinsed,counterstained with hematoxylin and mounted.

Clinical pathology was assessed by a pathologist.

Results

Mice were monitored weekly from 15 weeks old for proteinuriadevelopment. On week 16, mice were treated with 300 μg of Hamster Ig or8GB8 by i.p injection three times a week. To determine the effect of8GB8 on mediators in the plasma was collected and stored at −80° C.Spleens and lymph nodes were harvested and snap frozen forimmunofluorescence staining and RNA isolation. As shown in FIG. 74A-B8G8 reduces proteinuria development in MRL/Ipr mice. In theseexperiments 15 week old female MRL/Ipr mice were monitored weekly forproteinuria. Proteinuria values were recorded using chemstrips andquantified as mg/dL. On week 16, mice were treated with either 300 ughamster-Ig (black line, n=8) or 300 ug 8G8 (red line, n=8) by i.pinjection three times a week. Data at week 21 were discarded due totechnical problems with the chemstrips. (FIG. 74A) Average proteinuriais shown with standard error bars. (FIG. 74B) Disease incidence at eachtime point was calculated as the percent of mice in each group thatexhibited proteinuria at or greater than 100 mg/dL.

As further shown in FIG. 75 8G8 administration also reduces splenomegalyin MRL/Ipr mice. In these experiments spleens were harvested on week 23from mice were treated with either 300 ug Control-Ig/hamster-Ig or 300ug 8G8 by i.p injection three times a week. Splenomegaly was observed inControl-Ig treated mice compared to 8G8 treated mice. Shown here arerepresentative spleens.

As further shown in FIG. 76 8G8 administration also 8G8 reduceslymphoproliferation of cervical lymph nodes in MRL/pr mice. In theseexperiments cervical lymph nodes were harvested on week 23 from micetreated with either 300 ug Control-Ig/hamster-Ig or 300 ug 8G8 by i.pinjection three times a week. Lymphoproliferation was observed inControl-Ig treated mice compared to 8G8 treated mice.

Example 40: Effects of the α-Mouse VISTA Antibody 8G68 on Systemic LupusErythematosus—New Zealand Black x New Zealand White (NZBWF-1 Mice)

We examined the function of 8G8, a hamster α mouse VISTA antibody infemale NZBWF-1 mice. Mice were treated three times a week withcontrol-Ig or 8G8. Mice were monitored weekly for proteinuria and bodyweight. Serum was collected every two weeks during the treatment. At theend of the experiment, serum, spleens and kidneys were harvested. Serumwas stored at −80° C. until required for luminex assay.

Spleens were processed for flow cytometric analysis, cell sorting orsnap frozen in OCT for immunofluorescence staining and RNA isolation forgene profiling and nanoString analysis. One kidney was snap frozen inOCT for immunofluorescence staining and RNA isolation for gene profilingand nanoString analysis. The second kidney was fixed and paraffinembedded. Paraffin sections were H&E stained for clinical pathology.

Materials and Methods

Mouse Treatment

8-week old female NZBWF-1 mice were purchased from Jackson and stored inSPF conditions at the DHMC.

Proteinuria and body weight were monitored weekly in the DartmouthHitchcock animal facility.

Control-IgG/Hamster Ig or 8G8 was administered three times a week at 300μg/mouse by i.p injection

Mice were sacrificed upon signs of poor health and reduced activity, andaccording to animal facility protocols.

Proteinuria

Chemstrips 10 were purchased from Roche. Urine was collected from miceand placed onto the chemstrip. To determine protein in the urine, thecolorimetric scale was used: 0 mg/dL, trace (1 mg/dL), 30 mg/dL, 100mg/dL and 500 mg/dL.

Serum Analysis

Serum was collected and stored at −80° C. until required. Chemokine andcytokine levels were determined using a 32 Milliplex MouseCytokine/Chemokine Magnetic Bead Panel (Millipore) and the assays run ona Bio-plex 200 System (Life Science Research, Bio Rad). Data wasanalyzed using the Bio-Plex Manager 6.0 software.

Myeloid-Derived Suppressor Cell Isolation Kit

Myeloid-Derived Suppressor Cells (MDSCs) were isolated using theMyeloid-Derived Suppressor Cell Isolation Kit from Miltenyi Biotecaccording to the manufacturer's instructions.

RNA Isolation and nanoString

RNA was isolated from MDSCs using Trizol (Life Technologies) and thePureLink RNA Mini Kit (Ambion). RNA was run on a mouse inflammatorynanoString 12 assay (nanoString Technologies) and the data wasquantified using the nSolver Analysis Software.

Immunofluorescence Staining

Kidneys were embedded in OCT and 9 μl? sections cut and stored at −80°C. Slides were placed at RT for 20 mins, fixed in pre-chilled acetonefor 10 mins and removed to evaporate excess acetone, rehydrated in PBSfor 5 mins and sections circled using an ImmEdge pen (Vector Labs). Theywere incubated with 10% goat serum (Jackson ImmunoResearch) for 1 hr atRT and washed. Sections were stained with directly conjugated antibodiesto C3 and IgG diluted in PBS for 2 hr at RT and washed for 2 mins, thenmounted in ProLong Gold Antifade Mountant with DAPI (Life Technologies)and stored at RT in the dark for 48 hr. Images were acquired on a ZeissConfocal Microscope and analyzed with L5M 510 Meta software.

Clinical Pathology

Kidneys were placed into cassettes and fixed O/N in 10% Formalin at roomtemperature, then briefly washed in PBS and transferred and kept into70% Ethanol (Fisher Scientific) prior to being transferred to thePathology Translational Research Core at the Geisel School of Medicineat Dartmouth where they were paraffin embedded, sectioned and thenstained.

Paraffin embedded tissue sections (41 μm) were stained using a LeicaBOND RX automated stainer. After dewaxing, the sections were subjectedto antigen retrieval (Bond epitope retrieval solution 2, 100° C., 20min) and incubated with the primary antibody (see dilution below) for30-60 min, at room temperature in Leica diluent. Slides are then washed3×5 min washes in PBS and incubated with secondary antibody (from LeicaBond Refine detection kit, DS9800). After 3 final washes in PBS thesections were incubated with DAB (Leica Bond polymer detection kit),rinsed, counterstained with hematoxylin and mounted.

Clinical pathology was assessed by a pathologist.

Results

This experiment was designed to examine whether 8G8 mediated animmunosuppressive role in female NZBWF-1 mice. Mice were monitoredweekly from 22 weeks old for proteinuria development. On week 28, theweek after proteinuria was detected, mice were treated with 300 g ofHamster Ig or 8G8 by i.p injection three times a week. Whereas diseaseseverity in the control group continued to increase, the mice in the 8G8group displayed reduced proteinuria levels

Particularly, as shown in FIG. 77 8G8 antibody administration educesproteinuria development in NZBWF-1 mice. As shown therein these22-week-old female NZBWF-1 mice were monitored weekly for proteinuria.Proteinuria values were recorded using chemstrips and quantified asmg/dL. On week 28, mice were treated with either 300 ug Ham-Ig (blackline, n=6) or 300 ug 8G8 (red line, n=6) by i.p injection three times aweek.

As shown in FIG. 78 8G8 exerted no impact on immune complex depositionin NZBWF-1 mice. As shown therein the same 22-week-old female NZBWF-1mice were monitored weekly for proteinuria. Mice were treated 3 times aweek with 300 ug Ham-Ig (n=8) or 300 ug 8G8. To determine immune complex(ICs) deposition in B6, Ham-Ig and 8G8 mice, immunofluorescence stainingwas performed on frozen OCT kidney sections to detect C3 (red) and IgG(green) ICs by confocal microscopy at a magnification ×40 microscopy.

The results are further shown in the Table below. These values furthercorroborate that 8G8 reduces kidney damage in NZBWF-1 mice. In theexperiments clinical pathology was examined using paraffin embeddedkidneys from Ham-Ig or 8G8 treated NZBWF-1 and C57BL/6 (naïve controls)mice which were H&E stained and blindly clinically examined forinterstitial inflammation and glomerular damage. *Denotes significancebetween groups.

TABLE 4 8G8 reduces kidney damage in NZBWF-1 mice Interstitialinflammation Glomerular Strain score score B6 0 0 Ham-Ig 1.5 ± 0.5  2.5± 0.8  8G8 0.8 ± 0.4* 0.8 ± 0.4**

Conclusions

The results of these animal assays and other experiments disclosedherein indicate that agonist or immunosuppressive anti-VISTA agonistantibodies may be used for the treatment and prevention of autoimmune,allergic, inflammatory conditions or other conditions whereimmunosuppression is therapeutically desired; and in particular providecompelling evidence that agonist anti-VISTA agonist antibodies may beused for treating and preventing lupus, GVHD, RA, IBD, chronic infectionand hepatotoxicity, psoriasis and for preventing, reducing or managingthe symptoms of other acute and chronic autoimmune, allergic,inflammatory conditions.

REFERENCES CITED IN THIS APPLICATION

The following references and other references cited in this applicationare incorporated by reference in their entireties.

-   1 Dong, C., Juedes, A. E., Temann, U. A., Shresta, S., Allison, J.    P., Ruddle, N. H. and Flavell, R. A., ICOS co-stimulatory receptor    is essential for T-cell activation and function. Nature 2001. 409:    97-101.-   2 Suh, W. K., Gajewska, B. U., Okada, H., Gronski, M. A.,    Bertram, E. M., Dawicki, W., Duncan, G. S., Bukczynski, J., Plyte,    S., Elia, A., Wakeham, A., Itie, A., Chung, S., Da Costa, J., Arya,    S., Horan, T., Campbell, P., Gaida, K., Ohashi, P. S., Watts, T. H.,    Yoshinaga, S. K., Bray, M. R., Jordana, M. and Mak, T. W., The B7    family member B7-H3 preferentially down-regulates T helper type    1-mediated immune responses. Nat Immunol 2003. 4: 899-906.-   3 Borriello, F., Sethna, M. P., Boyd, S. D., Schweitzer, A. N.,    Tivol, E. A., Jacoby, D., Strom, T. B., Simpson, E. M.,    Freeman, G. J. and Sharpe, A. H., B7-1 and B7-2 have overlapping,    critical roles in immunoglobulin class switching and germinal center    formation. Immunity 1997. 6: 303-313.-   4 Chambers, C. A., Sullivan, T. J. and Allison, J. P.,    Lymphoproliferation in CTLA-4-deficient mice is mediated by    costimulation-dependent activation of CD4+ T cells. Immunity 1997.    7: 885-895.-   5 Waterhouse, P., Penninger, J. M., Timms, E., Wakeham, A.,    Shahinian, A., Lee, K. P., Thompson, C. B., Griesser, H. and Mak, T.    W., Lymphoproliferative disorders with early lethality in mice    deficient in Ctla-4. Science 1995. 270: 985-988.-   6 Tivol, E. A., Borriello, F., Schweitzer, A. N., Lynch, W. P.,    Bluestone, J. A. and Sharpe, A. H., Loss of CTLA-4 leads to massive    lymphoproliferation and fatal multiorgan tissue destruction,    revealing a critical negative regulatory role of CTLA-4.    Immunity 1995. 3: 541-547.-   7 Nishimura, H., Nose, M., Hiai, H., Minato, N. and Honjo, T.,    Development of lupus-like autoimmune diseases by disruption of the    PD-1 gene encoding an ITIM motif-carrying immunoreceptor.    Immunity 1999. 11: 141-151.-   8 Keir, M. E., Liang, S. C., Guleria, I., Latchman, Y. E., Qipo, A.,    Albacker, L. A., Koulmanda, M., Freeman, G. J., Sayegh, M. H. and    Sharpe, A. H., Tissue expression of PD-L1 mediates peripheral T cell    tolerance. J Exp Med 2006. 203: 883-895.-   9 Ortler, S., Leder, C., Mittelbronn, M., Zozulya, A. L., Knolle, P.    A., Chen, L., Kroner, A. and Wiendl, H., B7-H1 restricts    neuroantigen-specific T cell responses and confines inflammatory CNS    damage: implications for the lesion pathogenesis of multiple    sclerosis. Eur J Immunol 2008. 38: 1734-1744.-   10 Zhu, G., Augustine, M. M., Azuma, T., Luo, L., Yao, S., Anand,    S., Rietz, A. C., Huang, J., Xu, H., Flies, A. S., Flies, S. J.,    Tamada, K., Colonna, M., van Deursen, J. M. and Chen, L.,    B7-H4-deficient mice display augmented neutrophil-mediated innate    immunity. Blood 2009. 113: 1759-1767.-   11 Chen, Y., Wang, Q., Shi, B., Xu, P., Hu, Z., Bai, L. and Zhang,    X., Development of a sandwich ELISA for evaluating soluble PD-L1    (CD274) in human sera of different ages as well as supernatants of    PD-L1(+) cell lines. Cytokine 2011.-   12 Greenwald, R. J., Freeman, G. J. and Sharpe, A. H., The B7 family    revisited. Annu Rev Immunol 2005. 23: 515-548.-   13 Zhu, Y., Yao, S., Iliopoulou, B. P., Han, X., Augustine, M. M.,    Xu, H., Phennicie, R. T., Flies, S. J., Broadwater, M., Ruff, W.,    Taube, J. M., Zheng, L., Luo, L., Zhu, G., Chen, J. and Chen, L.,    B7-H5 costimulates human T cells via CD28H. Nat Commun 2013. 4:    2043.-   14 Brandt, C. S., Baratin, M., Yi, E. C., Kennedy, J., Gao, Z., Fox,    B., Haldeman, B., Ostrander, C. D., Kaifu, T., Chabannon, C.,    Moretta, A., West, R., Xu, W., Vivier, E. and Levin, S. D., The B7    family member B7-H6 is a tumor cell ligand for the activating    natural killer cell receptor NKp30 in humans. J Exp Med 2009. 206:    1495-1503.-   15 Wang, L., Rubinstein, R., Lines, J. L., Wasiuk, A., Ahonen, C.,    Guo, Y., Lu, L. F., Gondek, D., Wang, Y., Fava, R. A., Fiser, A.,    Almo, S. and Noelle, R. J., VISTA, a novel mouse Ig superfamily    ligand that negatively regulates T cell responses. J Exp Med 2011.    208: 577-592.-   16 Lines, J. L., Sempere, L. F., Wang, L., Panttazi, E., Mak, J.,    O'Connell, S., Ceeraz, S., Suriawinata, A. A., Yan, S.,    Ernstoff, M. S. and Noelle, R. J., VISTA is an immune checkpoint    regulator for human T cells. in revision (Cancer Research).-   17 LeMercier, I., Lines, J. L., Sergent, P., Li, J., Noelle, R. J.    and Wang, L., VISTA regulates the development of protective    anti-tumor immunity. in revision (Cancer Research).-   18 Wolchok, J. D., Kluger, H., Callahan, M. K., Postow, M. A.,    Rizvi, N. A., Lesokhin, A. M., Segal, N. H., Ariyan, C. E.,    Gordon, R. A., Reed, K., Burke, M. M., Caldwell, A., Kronenberg, S.    A., Agunwamba, B. U., Zhang, X., Lowy, I., Inzunza, H. D., Feely,    W., Horak, C. E., Hong, Q., Korman, A. J., Wigginton, J. M.,    Gupta, A. and Sznol, M., Nivolumab plus ipilimumab in advanced    melanoma. N Engl J Med 2013. 369: 122-133.-   19 Iliopoulos, D., Kavousanaki, M., loannou, M., Boumpas, D. and    Verginis, P., The negative costimulatory molecule PD-1 modulates the    balance between immunity and tolerance via miR-21. Eur J    Immunol 2011. 41: 1754-1763.-   20 Ansari, M. J., Salama, A. D., Chitnis, T., Smith, R. N., Yagita,    H., Akiba, H., Yamazaki, T., Azuma, M., Iwai, H., Khoury, S. J.,    Auchincloss, H., Jr. and Sayegh, M. H., The programmed death-1    (PD-1) pathway regulates autoimmune diabetes in nonobese diabetic    (NOD) mice. J Exp Med 2003. 198: 63-69.-   21 Bertsias, G. K., Nakou, M., Choulaki, C., Raptopoulou, A.,    Papadimitraki, E., Goulielmos, G., Kritikos, H., Sidiropoulos, P.,    Tzardi, M., Kardassis, D., Mamalaki, C. and Boumpas, D. T., Genetic,    immunologic, and immunohistochemical analysis of the programmed    death 1/programmed death ligand 1 pathway in human systemic lupus    erythematosus. Arthritis Rheum 2009. 60: 207-218.-   22 Prokunina, L., Castillejo-Lopez, C., Oberg, F., Gunnarsson, I.,    Berg, L., Magnusson, V., Brookes, A. J., Tentler, D.,    Kristjansdottir, H., Grondal, G., Bolstad, A. I., Svenungsson, E.,    Lundberg, I., Sturfelt, G., Jonssen, A., Truedsson, L., Lima, G.,    Alcocer-Varela, J., Jonsson, R., Gyllensten, U. B., Harley, J. B.,    Alarcon-Segovia, D., Steinsson, K. and Alarcon-Riquelme, M. E., A    regulatory polymorphism in PDCD1 is associated with susceptibility    to systemic lupus erythematosus in humans. Nat Genet 2002. 32:    666-669.-   23 Ozkaynak, E., Wang, L., Goodearl, A., McDonald, K., Qin, S.,    O'Keefe, T., Duong, T., Smith, T., Gutierrez-Ramos, J. C.,    Rottman, J. B., Coyle, A. J. and Hancock, W. W., Programmed death-1    targeting can promote allograft survival. J Immunol 2002. 169:    6546-6553.-   24 Watson, M. P., George, A. J. and Larkin, D. F., Differential    effects of costimulatory pathway modulation on corneal allograft    survival. Invest Ophthalmol Vis Sci 2006. 47: 3417-3422.-   25 Podojil, J. R., Liu, L. N., Marshall, S. A., Chiang, M. Y.,    Goings, G. E., Chen, L., Langermann, S. and Miller, S. D., B7-H4Ig    inhibits mouse and human T-cell function and treats EAE via    IL-10/Treg-dependent mechanisms. J Autoimmun 2013. 44: 71-81.-   26 Sica, G. L., Choi, I. H., Zhu, G., Tamada, K., Wang, S. D.,    Tamura, H., Chapoval, A. I., Flies, D. B., Bajorath, J. and Chen,    L., B7-H4, a molecule of the B7 family, negatively regulates T cell    immunity. Immunity 2003. 18: 849-861.-   27 Wang, X., Hao, J., Metzger, D. L., Mui, A., Ao, Z., Verchere, C.    B., Chen, L., Ou, D. and Warnock, G. L., B7-H4 induces    donor-specific tolerance in mouse islet allografts. Cell    Transplant 2012. 21: 99-111.-   28 Yamaura, K., Watanabe, T., Boenisch, O., Yeung, M., Yang, S.,    Magee, C. N., Padera, R., Datta, S., Schatton, T., Kamimura, Y.,    Azuma, M. and Najafian, N., In vivo function of immune inhibitory    molecule B7-H4 in alloimmune responses. Am J Transplant 2010. 10:    2355-2362.-   29 Yi, K. H. and Chen, L., Fine tuning the immune response through    B7-H3 and B7-H4. Immunol Rev 2009. 229: 145-151.-   30 Wang, X., Hao, J., Metzger, D. L., Ao, Z., Chen, L., Ou, D.,    Verchere, C. B., Mui, A. and Warnock, G. L., B7-H4 Treatment of T    Cells Inhibits ERK, JNK, p38, and AKT Activation. PLoS One 2012. 7:    e28232.-   31 Terawaki, S., Tanaka, Y., Nagakura, T., Hayashi, T., Shibayama,    S., Muroi, K., Okazaki, T., Mikami, B., Garboczi, D. N., Honjo, T.    and Minato, N., Specific and high-affinity binding of tetramerized    PD-L1 extracellular domain to PD-1-expressing cells: possible    application to enhance T cell function. Int Immunol 2007. 19:    881-890.-   32 Sedy, J. R., Gavrieli, M., Potter, K. G., Hurchla, M. A.,    Lindsley, R. C., Hildner, K., Scheu, S., Pfeffer, K., Ware, C. F.,    Murphy, T. L. and Murphy, K. M., B and T lymphocyte attenuator    regulates T cell activation through interaction with herpesvirus    entry mediator. Nat Immunol 2005. 6: 90-98.-   33 Parisi, S., Battista, M., Musto, A., Navarra, A., Tarantino, C.    and Russo, T., A regulatory loop involving Dies1 and miR-125a    controls BMP4 signaling in mouse embryonic stem cells. FASEB J 2012.    26: 3957-3968.-   34 Youngnak, P., Kozono, Y., Kozono, H., Iwai, H., Otsuki, N., Jin,    H., Omura, K., Yagita, H., Pardoll, D. M., Chen, L. and Azuma, M.,    Differential binding properties of B7-H1 and B7-DC to programmed    death-1. Biochem Biophys Res Commun 2003. 307: 672-677.-   35 Butte, M. J., Keir, M. E., Phamduy, T. B., Sharpe, A. H. and    Freeman, G. J., Programmed death-1 ligand 1 interacts specifically    with the b7-1 costimulatory molecule to inhibit T cell responses.    Immunity 2007. 27: 111-122.-   36 Sharpe, A. H. and Freeman, G. J., The B7-CD28 superfamily. Nat    Rev Immunol 2002. 2: 116-126.-   37 Bartel P. L. et al. (1993) Using the two-hybrid system to detect    protein-protein interactions. In Cellular Interactions in    Development: A Practical Approach, D. A. Hartley, Ed., Oxford    University Press, Oxford; pp 153-179.-   38 Béranger F. et al. (1997) Getting more from the two-hybrid    system: N-terminal fusions to LexA are efficient and sensitive baits    for two-hybrid studies. NAR 25: 2035-36.-   39 Formstecher E. et al. (2005) Protein interaction mapping: a    Drosophila case study.-   Genome Res. 15: 37684.-   40 Fromont-Racine M., Rain J. C., and Legrain P. (1997) Toward a    functional analysis of the yeast genome through exhaustive    two-hybrid screens. Nat. Genet. 16: 277-82.-   41 Rain J. C. et al. (2001) The protein-protein interaction map of    Helicobacter pylori. Nature 409: 211-15.-   42 Vojtek A. and Hollenberg S. M. (1995) Ras-Raf interaction:    two-hybrid analysis. Methods Enzymol. 255: 33142.-   43 Wojcik J., Boneca I. G., and Legrain P. (2002) Prediction,    assessment and validation of protein interaction maps in    bacteria. J. Mol. Biol. 323: 763-70.-   44. Franklin E C, Kunkel H G. Immunologic Differences Between the 19    S and 7 S Components of Normal Human γ-Globulin. The Journal of    Immunology. 1957; 78(1):11-8.-   45. Roda G, Jharap B, Neeraj N, Colombel J-F. Loss of Response to    Anti-TNFs: Definition, Epidemiology, and Management. Clin Trans    Gastroenterol. 2016; 7:e135. doi: 10.1038/ctg.2015.63.-   46. Greenwald R J, Freeman G J, Sharpe A H. The B7 family revisited.    Annual Review of Immunology. 2005; 23:515-48. PubMed PMID: 15771580.-   47. Lines J L, Pantazi E, Mak J, Sempere L F, Wang L, O'Connell S,    Ceeraz S, Suriawinata A A, Yan S, Ernstoff M S, Noelle R. VISTA is    an immune checkpoint molecule for human T cells. Cancer Research.    2014; 74(7):1924-32. doi: 10.1158/0008-5472.CAN-13-1504. PubMed    PMID: 24691993; PMCID: 3979527.-   48. Le Mercier I, Chen W, Lines J L, Day M, Li J, Sergent P, Noelle    R J, Wang L. VISTA Regulates the Development of Protective Antitumor    Immunity. Cancer Research. 2014; 74(7):1933-44. doi:    10.1158/0008-5472.CAN-13-1506. PubMed PMID: 24691994; PMCID:    PMC4116689.-   49. Flies D B, Han X, Higuchi T, Zheng L, Sun J, Ye J J, Chen L.    Coinhibitory receptor PD-1H preferentially suppresses CD4(+) T    cell-mediated immunity. The Journal of Clinical Investigation. 2014;    124(5):1966-75. doi: 10.1172/JCI74589. PubMed PMID: 24743150; PMCID:    4001557.-   50. Lines J L, Sempere L F, Broughton T, Wang L, Noelle R. VISTA Is    a Novel Broad-Spectrum Negative Checkpoint Regulator for Cancer    Immunotherapy. Cancer Immunology research. 2014; 2(6):510-7. doi:    10.1158/2326-6066.CIR-14-0072. PubMed PMID: 24894088.-   51. Wang L, Le Mercier I, Putra i, Chen W, Liu J, Schenk A D, Nowak    E C, Suriawinata A A, Li J, Noelle R J. Disruption of the    immune-checkpoint VISTA gene imparts a proinflammatory phenotype    with predisposition to the development of autoimmunity. Proceedings    of the National Academy of Sciences of the United States of America.    2014; 111(41):14846-51. doi: 10.1073/pnas.1407447111. PubMed PMID:    25267631; PMCID: 4205642.-   52. Wang L, Rubinstein R, Lines J L, Wasiuk A, Ahonen C, Guo Y, Lu L    F, Gondek D, Wang Y, Fava R A, Fiser A, Almo S, Noelle R J. VISTA, a    novel mouse Ig superfamily ligand that negatively regulates T cell    responses. The Journal of Experimental Medicine. 2011;    208(3):577-92. Epub 2011/03/09. doi: jem.20100619 [pii]    10.1084/jem.20100619. PubMed PMID: 21383057; PMCID: 3058578.-   53. Flies D B, Han X, Higuchi T, Zheng L, Sun J, Ye J J, Chen L.    Coinhibitory receptor PD-1H preferentially suppresses CD4(+) T    cell-mediated immunity. The Journal of Clinical Investigation. 2014;    124(5):1966-75. doi: 10.1172/JC174589. PubMed PMID: PMC4001557.-   54. Yoon K W, Byun S, Kwon E, Hwang S Y, Chu K, Hiraki M, Jo S H,    Weins A, Hakroush S, Cebulla A, Sykes D B, Greka A, Mundel P, Fisher    D E, Mandinova A, Lee S W. Control of signaling-mediated clearance    of apoptotic cells by the tumor suppressor p53. Science. 2015;    349(6247):1261669. doi: 10.1126/science.1261669. PubMed PMID:    26228159.-   55. Bettelli E, Pagany M, Weiner H L, Linington C, Sobel R A,    Kuchroo V K. Myelin Oligodendrocyte Glycoprotein-specific T Cell    Receptor Transgenic Mice Develop Spontaneous Autoimmune Optic    Neuritis. The Journal of Experimental Medicine. 2003;    197(9):1073-81. doi: 10.1084/jem.20021603.-   56. Ceeraz S, Sergent P, Plummer S, Schned A, Pechenick D, Burns C,    Noelle R. VISTA deficiency accelerates the development of fatal    murine lupus nephritis. Arthritis and Rheumatology. 2016; submitted.-   57. Liu J, Yuan Y, Chen W, Putra J, Suriawinata A A, Schenk A D,    Miller H E, Guleria I, Barth R J, Huang Y H, Wang L.    Immune-checkpoint proteins VISTA and PD-1 nonredundantly regulate    murine T-cell responses. Proceedings of the National Academy of    Sciences of the United States of America. 2015; 112(21):6682-7. doi:    10.1073/pnas.1420370112. PubMed PMID: 25964334; PMCID: PMC4450438.-   58. Flies D B, Higuchi T, Chen L. Mechanistic Assessment of PD-1H    Coinhibitory Receptor-Induced T Cell Tolerance to Allogeneic    Antigens. Journal of Immunology. 2015; 194(11):5294-304. doi:    10.4049/jimmunol.1402648. PubMed PMID: 25917101; PMCID: PMC4433880.-   59. DiLillo D J, Ravetch J V. Fc-Receptor Interactions Regulate Both    Cytotoxic and Immunomodulatory Therapeutic Antibody Effector    Functions. Cancer Immunology Research. 2015; 3(7):704-13. doi:    10.1158/2326-6066.cir-15-0120.-   60. White A L, Chan H T, French R R, Willoughby J, Mockridge C I,    Roghanian A, Penfold C A, Booth S G, Dodhy A, Polak M E, Potter E A,    Ardern-Jones M R, Verbeek J S, Johnson P W, Al-Shamkhani A, Cragg M    S, Beers S A, Glennie M J. Conformation of the human immunoglobulin    G2 hinge imparts superagonistic properties to immunostimulatory    anticancer antibodies. Cancer Cell. 2015; 27(1):138-48. doi:    10.1016/j.ccell.2014.11.001. PubMed PMID: 25500122; PMCID:    PMC4297290.-   61. Dubey A K, Handu S S, Dubey S, Sharma P, Sharma K K, Ahmed Q M.    Belimumab: First targeted biological treatment for systemic lupus    erythematosus. J Pharmacol Pharmacother. 2011; 2(4):317-9. doi:    10.4103/0976-500X.85930. PubMed PMID: 22025872; PMCID: PMC3198539.-   62. Wallace D J, Hobbs K, Clowse M E, Petri M, Strand V, Pike M,    Merrill J T, Leszczynski P, Neuwelt C M, Jeka S, Houssiau F,    Keiserman M, Ordi-Ros J, Bongardt S, Kilgallen B, Galateanu C,    Kalunian K, Furie R, Gordon C. Long-term safety and efficacy of    epratuzumab in the treatment of moderate-to-severe systemic lupus    erythematosus: results from an open-label extension study. Arthritis    Care Res (Hoboken). 2015. doi: 10.1002/acr.22694. PubMed PMID:    26316325.-   63. Van Wauwe J P, De Mey J R, Goossens J G. OKT3: a monoclonal    anti-human T lymphocyte antibody with potent mitogenic properties.    The Journal of Immunology. 1980; 124(6):2708-13.-   64. Robertson J M, Jensen P E, Evavold B D. DO11.10 and OT-II T    Cells Recognize a C-Terminal Ovalbumin 323-339 Epitope. The Journal    of Immunology. 2000; 164(9):4706-12. doi:    10.4049/jimmunol.164.9.4706.-   65. Wang H-X, Liu M, Weng S-Y, Li J-J, Xie C, He H-L, Guan W, Yuan    Y-S, Gao J. Immune mechanisms of Concanavalin A model of autoimmune    hepatitis. World Journal of Gastroenterology: WJG. 2012;    18(2):119-25. doi: 10.3748/wjg.v18.i2.119. PubMed PMID: PMC3257438.-   66. Weiner G J. Building better monoclonal antibody-based    therapeutics. Nat Rev Cancer. 2015; 15(6):361-70. doi:    10.1038/nrc3930. PubMed PMID: 25998715; PMCID: PMC4491443.-   67. Ravetch J V, Bolland S. IgG Fc receptors. Annual Review of    Immunology. 2001; 19:275-90. doi: 10.1146/annurev.immunol.19.1.275.    PubMed PMID: 11244038.-   68. Li F, Smith P, Ravetch J V. Inhibitory Fcγ receptor is required    for the maintenance of tolerance through distinct mechanisms.    Journal of Immunology (Baltimore, Md.: 1950). 2014; 192(7):3021-8.    doi: 10.4049/jimmunol.1302934. PubMed PMID: PMC3967505.-   69. Hinton P R, Johlfs M G, Xiong J M, Hanestad K, Ong K C, Bullock    C, Keller S, Tang M T, Tso J Y, Vasquez M, Tsurushita N. Engineered    human IgG antibodies with longer serum half-lives in primates. The    Journal of Biological Chemistry. 2004; 279(8):6213-6. doi:    10.1074/jbc.C300470200. PubMed PMID: 14699147.-   70. Vaccaro C, Zhou J, Ober R J, Ward E S. Engineering the Fc region    of immunoglobulin G to modulate in vivo antibody levels. Nature    Biotechnology. 2005; 23(10):1283-8. doi: 10.1038/nbt1143. PubMed    PMID: 16186811.-   71. Borrok M J, Wu Y, Beyaz N, Yu X-Q, Oganesyan V, Dall'Acqua W F,    Tsui P. pH-dependent Binding Engineering Reveals an FcRn Affinity    Threshold That Governs IgG Recycling. The Journal of Biological    Chemistry. 2015; 290(7):4282-90. doi: 10.1074/jbc.M114.603712.    PubMed PMID: PMC4326836.-   72. Oyarzun P, Ellis J J, Gonzalez-Galarza F F, Jones A R, Middleton    D, Boden M, Kobe B. A bioinformatics tool for epitope-based vaccine    design that accounts for human ethnic diversity: Application to    emerging infectious diseases. Vaccine. 2015; 33(10):1267-73. doi:    http://dx.doi.org/10.1016/j.vaccine.2015.01.040.-   73. Haskins K, Kubo R, White i, Pigeon M, Kappler J, Marrack P. The    major histocompatibility complex-restricted antigen receptor on T    cells. I. Isolation with a monoclonal antibody. The Journal of    Experimental Medicine. 1983; 157(4):1149-69. Epub 1983/04/01. PubMed    PMID: 6601175; PMCID: Pmc2186983.-   74. Markees T G, Phillips N E, Noelle R J, Shultz L D, Mordes J P,    Greiner D L, Rossini A A. Prolonged survival of mouse skin    allografts in recipients treated with donor splenocytes and antibody    to CD40 ligand. Transplantation. 1997; 64(2):329-35.-   75. Ehst B D, Ingulli E, Jenkins M K. Development of a novel    transgenic mouse for the study of interactions between CD4 and CD8 T    cells during graft rejection. American Journal of Transplantation:    Official journal of the American Society of Transplantation and the    American Society of Transplant Surgeons. 2003; 3(11):1355-62. PubMed    PMID: 14525595.-   76. Wu S, Jin L, Vence L, Radvanyi L G. Development and application    of ‘phosphoflow’ as a tool for immunomonitoring. Expert Rev    Vaccines. 2010; 9(6):631-43. doi: 10.1586/erv.10.59. PubMed PMID:    20518718; PMCID: PMC2933839.-   77. Weissmuller S, Kronhart S, Kreuz D, Schnierle B, Kalinke U,    Kirberg J, Hanschmann K M, Waibler Z. TGN1412 Induces Lymphopenia    and Human Cytokine Release in a Humanized Mouse Model. PloS One.    2016; 11(3):e0149093. doi: 10.1371/journal.pone.0149093. PubMed    PMID: 26959227; PMCID: PMC4784892.-   78. Piccotti J R, Alvey J D, Reindel J F, Guzman R E.    T-cell-dependent antibody response: assay development in cynomolgus    monkeys. J Immunotoxicol. 2005; 2(4):191-6. doi:    10.1080/15476910500362838. PubMed PMID: 18958673.-   79. Muller P Y, Brennan F R. Safety assessment and dose selection    for first-in-human clinical trials with immunomodulatory monoclonal    antibodies. Clin Pharmacol Ther. 2009; 85(3):247-58. doi:    10.1038/clpt.2008.273. PubMed PMID: 19177065

SEQUENCE LISTINGSEQ ID NO: 1: Homo sapiens VISTA (Alternate names: B7-H5; B7H5; DD1alpha; GI24; PP2135;SISP1) AMINO ACID SEQUENCE   1mgvptaleag swrwgsllfa lflaaslgpv aafkvatpys lyvcpegqnv tltcrllgpv  61dkghdvtfyk twyrssrgev qtcserrpir nltfqdlhlh hgghqaants hdlaqrhgle 121sasdhhgnfs itmrnltlld sglycclvve irhhhsehrv hgamelqvqt gkdapsncvv 181ypsssqdsen itaaalatga civgilclpl illlvykqrq aasnrraqel vrmdsniqgi 241enpgfeaspp aqgipeakvr hplsyvaqrq psesgrhlls epstplsppg pgdvffpsld 301pvpdspnfev i SEQ ID NO: 2: Mus musculus VISTA AMINO ACID SEQUENCE   1mgvpavpeas sprwgtllla iflaasrglv aafkvttpys lyvcpegqna tltcrilgpv  61skghdvtiyk twylssrgev qmckehrpir nftlqhlqhh gshlkanash dqpqkhglel 121asdhhgnfsi tlrnvtprds glycclviel knhhpeqrfy gsmelqvqag kgsgstcmas 181neqdsdsita aalatgaciv gilclplill lvykqrqvas hrraqelvrm dsntqgienp 241gfettppfqg mpeaktrppl syvaqrqpse sgryllsdps tplsppgpgd vffpsldpvp 301dspnseai SEQ ID NO: 3: Mus musculus VISTA AMINO ACID SEQUENCE   1mgvpavpeas sprwgtllla iflaasrglv aafkvttpys lyvcpegqna tltcrilgpv  61skghdvtiyk twylssrgev qmckehrpir nftlqhlqhh gshlkanash dqpqkhglel 121asdhhgnfsi tlrnvtprds glycclviel knhhpeqrfy gsmelqvqag kgsgstcmas 181neqdsdsita aalatgaciv gilclplill lvykqrqvas hrraqelvrm dssntqgien 241pgfettppfq gmpeaktrpp lsyvaqrqps esgryllsdp stplsppgpg dvffpsldpv 301pdspnseaiSEQ ID NO: 4: Homo sapiens VISTA (Alternate names: B7-H5; B7H5; DD1alpha; GI24; PP2135;SISP1) NUCLEIC ACID SEQUENCE    1gggggcgggt gcctggagca cggcgctggg gccgcccgca gcgctcactc gctcgcactc   61agtcgcggga ggcttccccg cgccggccgc gtcccgcccg ctuccggca ccagaagttc  121ctctgcgcgt ccgacggcga catgggcgtc cccacggccc tggaggccgg cagctggcgc  181tggggatccc tgctcttcgc tctcttcctg gctgcgtccc taggtccggt ggcagccttc  241aaggtcgcca cgccgtattc cctgtatgtc tgtcccgagg ggcagaacgt caccctcacc  301tgcaggctct tgggccctgt ggacaaaggg cacgatgtga ccttctacaa gacgtggtac  361cgcagctcga ggggcgaggt gcagacctgc tcagagcgcc ggcccatccg caacctcacg  421ttccaggacc ttcacctgca ccatggaggc caccaggctg ccaacaccag ccacgacctg  481gctcagcgcc acgggctgga gtcggcctcc gaccaccatg gcaacttctc catcaccatg  541cgcaacctga ccctgctgga tagcggcctc tactgctgcc tggtggtgga gatcaggcac  601caccactcgg agcacagggt ccatggtgcc atggagctgc aggtgcagac aggcaaagat  661gcaccatcca actgtgtggt gtacccatcc tcctcccagg atagtgaaaa catcacggct  721gcagccctgg ctacgggtgc ctgcatcgta ggaatcctct gcctccccct catcctgctc  781ctggtctaca agcaaaggca ggcagcctcc aaccgccgtg cccaggagct ggtgcggatg  841gacagcaaca ttcaagggat tgaaaacccc ggctttgaag cctcaccacc tgcccagggg  901atacccgagg ccaaagtcag gcaccccctg tcctatgtgg cccagcggca gccttctgag  961tctgggcggc atctgctttc ggagcccagc acccccctgt ctcctccagg ccccggagac 1021gtcttcttcc catccctgga ccctgtccct gactctccaa actttgaggt catctagccc 1081agctggggga cagtgggctg ttgtggctgg gtctggggca ggtgcatttg agccagggct 1141ggctctgtga gtggcctcct tggcctcggc cctggttccc tccctcctgc tctgggctca 1201gatactgtga catcccagaa gcccagcccc tcaacccctc tggatgctac atggggatgc 1261tggacggctc agcccctgtt ccaaggattt tggggtgctg agattctccc ctagagacct 1321gaaattcacc agctacagat gccaaatgac ttacatctta agaagtctca gaacgtccag 1381cccttcagca gctctcgttc tgagacatga gccttgggat gtggcagcat cagtgggaca 1441agatggacac tgggccaccc tcccaggcac cagacacagg gcacggtgga gagacttctc 1501ccccgtggcc gccttggctc ccccgttttg cccgaggctg ctcttctgtc agacttcctc 1561tttgtaccac agtggctctg gggccaggcc tgcctgccca ctggccatcg ccaccttccc 1621cagctgcctc ctaccagcag tttctctgaa gatctgtcaa caggttaagt caatctgggg 1681cttccactgc ctgcattcca gtccccagag cttggtggtc ccgaaacggg aagtacatat 1741tggggcatgg tggcctccgt gagcaaatgg tgtcttgggc aatctgaggc caggacagat 1801gttgccccac ccactggaga tggtgctgag ggaggtgggt ggggccttct gggaaggtga 1861gtggagaggg gcacctgccc cccgccctcc ccatccccta ctcccactgc tcagcgcggg 1921ccattgcaag ggtgccacac aatgtcttgt ccaccctggg acacttctga gtatgaagcg 1981ggatgctatt aaaaactaca tggggaaaca ggtgcaaacc ctggagatgg attgtaagag 2041ccagtttaaa tctgcactct gctgctcctc ccccaccccc accttccact ccatacaatc 2101tgggcctggt ggagtcttcg cttcagagcc attcggccag gtgcgggtga tgttcccatc 2161tcctgcttgt gggcatgccc tggctttgtt tttatacaca taggcaaggt gagtcctctg 2221tggaattgtg attgaaggat tttaaagcag gggaggagag tagggggcat ctctgtacac 2281tctgggggta aaacagggaa ggcagtgcct gagcatgggg acaggtgagg tggggctggg 2341cagaccccct gtagcgttta gcaggatggg ggccccaggt actgtggaga gcatagtcca 2401gcctgggcat ttgtctccta gcagcctaca ctggctctgc tgagctgggc ctgggtgctg 2461aaagccagga tttggggcta ggcgggaaga tgttcgccca attgcttggg gggttggggg 2521gatggaaaag gggagcacct ctaggctgcc tggcagcagt gagccctggg cctgtggcta 2581cagccaggga accccacctg gacacatggc cctgcttcta agccccccag ttaggcccaa 2641aggaatggtc cactgagggc ctcctgctct gcctgggctg ggccaggggc tttgaggaga 2701gggtaaacat aggcccggag atggggctga cacctcgagt ggccagaata tgcccaaacc 2761ccggcttctc ccttgtccct aggcagaggg gggtcccttc ttttgttccc tctggtcacc 2821acaatgcttg atgccagctg ccataggaag agggtgctgg ctggccatgg tggcacacac 2881ctgtcctccc agcactttgc agggctgagg tggaaggacc gcttaagccc aggtgttcaa 2941ggctgctgtg agctgtgttc gagccactac actccagcct ggggacggag caaaactttg 3001cctcaaaaca aattttaaaa agaaagaaag aaggaaagag ggtatgtttt tcacaattca 3061tgggggcctg catggcagga gtggggacag gacacctgct gttcctggag tcgaaggaca 3121agcccacagc ccagattccg gttctcccaa ctcaggaaga gcatgccctg ccctctgggg 3181aggctggcct ggccccagcc ctcagctgct gaccttgagg cagagacaac ttctaagaat 3241ttggctgcca gaccccaggc ctggctgctg ctgtgtggag agggaggcgg cccgcagcag 3301aacagccacc gcacttcctc ctcagcttcc tctggtgcgg ccctgccctc tcttctctgg 3361acccttttac aactgaacgc atctgggctt cgtggtttcc tgttttcagc gaaatttact 3421ctgagctccc agttccatct tcatccatgg ccacaggccc tgcctacaac gcactaggga 3481cgtccctccc tgctgctgct ggggaggggc aggctgctgg agccgccctc tgagttgccc 3541gggatggtag tgcctctgat gccagccctg gtggctgtgg gctggggtgc atgggagagc 3601tgggtgcgag aacatggcgc ctccaggggg cgggaggagc actaggggct ggggcaggag 3661gctcctggag cgctggattc gtggcacagt ctgaggccct gagagggaaa tccatgcttt 3721taagaactaa ttcattgtta ggagatcaat caggaattag gggccatctt acctatctcc 3781tgacattcac agtttaatag agacttcctg cctttattcc ctcccaggga gaggctgaag 3841gaatggaatt gaaagcacca tttggagggt tttgctgaca cagcggggac tgctcagcac 3901tccctaaaaa cacaccatgg aggccactgg tgactgctgg tgggcaggct ggccctgcct 3961gggggagtcc gtggcgatgg gcgctggggt ggaggtgcag gagccccagg acctgctttt 4021caaaagactt ctgcctgacc agagctccca ctacatgcag tggcccaggg cagaggggct 4081gatacatggc ctttttcagg gggtgctcct cgcggggtgg acttgggagt gtgcagtggg 4141acagggggct gcaggggtcc tgccaccacc gagcaccaac ttggcccctg gggtcctgcc 4201tcatgaatga ggccttcccc agggctggcc tgactgtgct gggggctggg ttaacgtttt 4261ctcagggaac cacaatgcac gaaagaggaa ctggggttgc taaccaggat gctgggaaca 4321aaggcctctt gaagcccagc cacagcccag ctgagcatga ggcccagccc atagacggca 4381caggccacct ggcccattcc ctgggcattc cctgctttgc attgctgctt ctcttcaccc 4441catggaggct atgtcaccct aactatcctg gaatgtgttg agagggattc tgaatgatca 4501atatagcttg gtgagacagt gccgagatag atagccatgt ctgccttggg cacgggagag 4561ggaagtggca gcatgcatgc tgtttcttgg ccttttctgt tagaatactt ggtgctttcc 4621aacacacttt cacatgtgtt gtaacttgtt tgatccaccc ccttccctga aaatcctggg 4681aggttttatt gctgccattt aacacagagg gcaatagagg ttctgaaagg tctgtgtctt 4741gtcaaaacaa gtaaacggtg gaactacgac taaa //  SEQ ID NO: 5: Homo sapiens VISTA (Alternate names: B7-H5; B7H5; DD1alpha; GI24; PP2135;SISP1) CODING NUCLEIC ACID SEQUENCE    1ctcgccgcgc tgagccgcct cgggacggag ccatgcggcg ctgggcctgg gccgcggtcg   61tggtccccct cgggccgcag ctcgtgctcc tcgggggcgt cggggcccgg cgggaggcac  121agaggacgca gcagcctggc cagcgcgcag atccccccaa cgccaccgcc agcgcgtcct  181cccgcgaggg gctgcccgag gcccccaagc catcccaggc ctcaggacct gagttctccg  241acgcccacat gacatggctg aactttgtcc ggcggccgga cgacggcgcc ttaaggaagc  301ggtgcggaag cagggacaag aagccgcggg atctcttcgg tttcccagga cctccaggtg  361cagaagtgac cgcggagact ctgcttcacg agtttcagga gctgctgaaa gaggccacgg  421agcgccggtt ctcagggctt ctggacccgc tgctgcccca gggggcgggc ctgcggctgg  481tgggcgaggc ctttcactgc cggctgcagg gtccccgccg ggtggacaag cggacgctgg  541tggagctgca tggtttccag gctcctgctg cccaaggtgc cttcctgcga ggctccggtc  601tgagcctggc ctcgggtcgg ttcacggccc ccgtgtccgg catcttccag ttctctgcca  661gtctgcacgt ggaccacagt gagctgcagg gcaaggcccg gctgcgggcc cgggacgtgg  721tgtgtgttct catctgtatt gagtccctgt gccagcgcca cacgtgcctg gaggccgtct  781caggcctgga gagcaacagc agggtcttca cgctacaggt gcaggggctg ctgcagctgc  841aggctggaca gtacgcttct gtgtttgtgg acaatggctc cggggccgtc ctcaccatcc  901aggcgggctc cagcttctcc gggctgctcc tgggcacgtg agggcgccca ggggggctgg  961cgaggagctg ccgccggatc ccggggaccc tcctactgat gcccgtggtc accacaataa 1021agagccctcc accctcaaaa aaaaaaaaaa aaaaa //SEQ ID NO: 6: Mus musculus VISTA CODING NUCLEIC ACID SEQUENCE    1ctcgccgcgc tgagccgcct cgggacggag ccatgcggcg ctgggcctgg gccgcggtcg   61tggtccttct cgggccgcag ctcgtgctcc tcgggggcgt cggggcccgg cgggaggcac  121agaggacgca gcagcctggc cagcgcgcag atccccccaa cgccaccgcc agcgcgtcct  181cccgcgaggg gctgcccgag gcccccaagc catcccaggc ctcaggacct gagttctccg  241acgcccacat gacatggctg aactttgtcc ggcggccgga cgacggcgcc ttaaggaagc  301ggtgcggaag cagggacaag aagccgcggg atctcttcgg tttcccagga cctccaggtg  361cagaagtgac cgcggagact ctgcttcacg agtttcagga gctgctgaaa gaggccacgg  421agcgccggtt ctcagggctt ctggacccgc tgctgcccca gggggcgggc ctgcggctgg  481tgggcgaggc ctttcactgc cggctgcagg gtccccgccg ggtggacaag cggacgctgg  541tggagctgca tggtttccag gctcctgctg cccaaggtgc cttcctgcga ggctttggtc  601tgagcctggc ctcgggtcgg ttcacggccc ccgtgtccgg catcttccag ttctctgcca  661gtctgcacgt ggaccacagt gagctgcagg gcaaggcccg gctgcgggcc cgggacgtgg  721tgtgtgttct catctgtatt gagtccctgt gccagcgcca cacgtgcctg gaggccgtct  781caggcctgga gagcaacagc agggtcttca cgctacaggt gcaggggctg ctgcagctgc  841aggctggaca gtacgcttct gtgtttgtgg acaatggctc cggggccgtc ctcaccatcc  901aggcgggctc cagcttctcc gggctgctcc tgggcacgtg agggcgccca ggggggctgg  961cgaggagctg ccgccggatc ccggggaccc tcctactgat gcccgtggtc accacaataa  1021agagccctcc accctcaaaa aaaaaaaaaa aaaaa //

1. A method of treating or preventing an autoimmune, allergic orinflammatory condition comprising administering to a patient in needthereof a therapeutically or prophylactically effective amount of anisolated antibody or antibody fragment thereof comprising an antigenbinding region that specifically binds to human V-domain Ig Suppressorof T cell Activation (human VISTA), wherein the antibody or antibodyfragment agonizes or promotes one or more of the effects of VISTA onimmunity. 2-119. (canceled)
 120. A method of treating or preventing aninflammatory bowel or gastrointestinal condition including but notlimited to IBD or colitis comprising administering to a patient in needthereof a therapeutically or prophylactically effective amount of anisolated antibody or antibody fragment thereof comprising an antigenbinding region that specifically binds to human V-domain Ig Suppressorof T cell Activation (human VISTA), wherein the antibody or antibodyfragment agonizes or promotes one or more of the effects of VISTA onimmunity
 121. The method of claim 120, wherein the antibody comprises ahuman IgG2 constant or human IgG2 Fc region.
 122. The method of claim120, wherein the human IgG2 constant or Fc region of the antibody bindsto Fc gamma receptors including human CD32A.
 123. The method of claim120, wherein the IgG2 constant or Fc region comprises the native humanIgG2 binding to Fc gamma receptors.
 124. The method of claim 123,wherein said FcyRs include one or more of hFcγRI(CD64), FcyRIIA orhFcyRIIB, (CD32 or CD32A) and FcγR111A (CD16A) or FcγR111B (CD16B). 125.The method of claim 120 wherein the isolated antibody or antibodyfragment competes with or binds to a VISTA epitope which includes oroverlaps with the epitope bound by any of the anti-human VISTAantibodies having the sequences of FIG.
 4. 126. The method of claim 125wherein the isolated antibody or antibody fragment binds or interactswith one of more residues of an epitope comprising residues ofLLDSGLYCCLVVEIRHHHSEHRVH(SEQ ID NO:92).
 127. The method of claim 125wherein the isolated antibody or antibody fragment binds or interactswith one of more residues of an epitope comprising one or more residuesof ₇₉EVQTCSERRPIR₉₀ (SEQ ID NO:68), ₄₈NVTLTCRLLGPV₆₀,₁₅₃HHHSEHRVHGAM₁₆₄, ₅₂LTCRLLGPV₆₀, ₅₆LLGPVDKGHDVTFYK₇₀,₁₁₃LAQRHGLESASDHHG₁₂₇, ₁₅₃HHHSEHRVHGAM₁₆₄, ₉₃TFQDLHLHHGGHQAA₁₀₇,₁₄₆CLVVEIRHHHSEH₁₅₈, ₅₃TCRLLGPVDKG₆₃, ₁₂₃SDHHG₁₂₇ and/or₁₅₃HHHSEHRVHGAM₁₆₄.
 128. The method of claim 125 wherein the isolatedantibody or antibody fragment binds or interacts with one of moreresidues of an epitope comprising one or more residues of₇₉EVQTCSERRPIR₉₀ (SEQ ID NO:68).
 129. The method of claim 120 whereinthe isolated antibody or antibody fragment promotes or enhances at leastone effect of human VISTA on immunity, e.g. its suppressive effect onany one or more of T cell immunity, activation of monocytes, inductionof T-cell proliferation; induction or suppression of cytokineexpression, increased survival of monocytes, induction ofantibody-dependent cell-mediated cytotoxicity (ADCC) in cells-expressingVISTA; and induction of antibody-dependent cellular phagocytosis (ADCP)in cells-expressing VISTA.
 130. The method of claim 120 wherein theisolated antibody or antibody fragment comprises an antigen bindingregion that specifically binds to human VISTA, wherein the antibody orantibody fragment comprises variable heavy and light sequences havingthe identical CDR polypeptides as any one of the anti-human VISTAantibodies having the CDR and variable heavy and light polypeptidesshown in FIG.
 4. 131. The method of claim 120 wherein the isolatedantibody or antibody fragment comprises the same CDRs as an antibodyselected from VSTB49-VSTB116.
 132. The method of claim 120 wherein theisolated antibody or antibody fragment comprises a variable heavy and/orvariable light polypeptide having at least 90% sequence identity tothose of an anti-human VISTA antibody selected from any one ofVSTB49-VSTB116, wherein the variable heavy and light polypeptidesequences thereof are shown in FIG.
 4. 133. The method of claim 120wherein the isolated antibody or antibody fragment comprises a variableheavy and/or variable light polypeptide having at least 95% sequenceidentity to those of an anti-human VISTA antibody selected from any oneof VSTB49-VSTB116, wherein the variable heavy and light polypeptidesequences thereof are shown in FIG.
 4. 134. The method of claim 120wherein the isolated antibody or antibody fragment comprises a variableheavy and/or variable light polypeptide having at least 96-99% sequenceidentity to those of an anti-human VISTA antibody selected from any oneof VSTB49-VSTB116.
 135. The method of claim 120 wherein the isolatedantibody or antibody fragment comprises a variable heavy and/or variablelight polypeptide identical to those of an anti-human VISTA antibodyselected from one of VSTB49-VSTB116, wherein the variable heavy andlight polypeptide sequences thereof are shown in FIG.
 4. 136. The methodof claim 120 wherein the isolated antibody or antibody fragmentcomprises a human constant domain.
 137. The method of claim 120 whereinthe isolated antibody or antibody fragment comprises wherein theantibody fragment comprises or is a Fab, F(ab′)2, or scFv antibodyfragment.
 138. The method of claim 120 wherein the isolated antibody orantibody fragment comprises an agonistic anti-human VISTA antibody orantibody fragment which promotes or enhances at least one of the effectsof human VISTA on immunity, e.g., selected from its suppressive effect Tcell immunity, activation of monocytes, suppression of T-cellproliferation; induction or suppression of cytokine expression,increased survival of monocytes, suppression of antibody-dependentcell-mediated cytotoxicity (ADCC) in cells-expressing VISTA; andsuppression of antibody-dependent cellular phagocytosis (ADCP) ofcells-expressing VISTA.